Cell culture models for validating candidate compounds for use in treating COPD and other diseases

ABSTRACT

The present invention relates to methods of diagnosing, monitoring, and treating elastin fiber injuries. In additional preferred embodiments, the present invention relates to methods of validating candidate compounds for use in treating chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, refractory asthma, and other related diseases. Examples of such methods include determining if the candidate compound decreases the degradation of elastic fiber in a patient administered the candidate compound by measuring, using mass spectrometry employing an internal standard, a marker of elastic fiber degradation in a sample of a body fluid or a tissue of the patient. The invention provides that a decrease in the presence of the marker compared to a control validates that the candidate compound is effective to treat, prevent, or ameliorate the disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 13/836,332, filed Mar. 15, 2013, now U.S. Pat. No.9,068,942, issued Jun. 30, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/575,346, now abandoned, which is the nationalstage of International Application No. PCT/US2011/022619, filed Jan. 26,2011, which claims benefit to U.S. provisional application Ser. No.61/336,804 filed Jan. 26, 2010, the entire contents of which areincorporated by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrently herewith as sequence listingtext file COPD_ST25.txt, file size 16.7 KB, created on May 13, 2015. Theaforementioned sequence listing is hereby incorporated by reference inits entirety pursuant to 37 C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to the field of elastin fiber injuriesand, more particularly, to methods of diagnosing, monitoring, andtreating elastin fiber injuries. Still further, the present inventionrelates to methods of validating candidate compounds for use in treatingelastin fiber injuries, such as those injuries caused by chronicobstructive pulmonary disease (COPD), chronic bronchitis, emphysema,refractory asthma, and other related diseases.

BACKGROUND OF THE INVENTION

Lung elastin degradation occurs with the development of pulmonaryemphysema in patients with COPD related to smoking or alpha-1antitrypsin deficiency. COPD a lengthy, chronic, progressive disease, isdebilitating and often non-reversible. COPD is a growing and costlyproblem. COPD currently affects over 18 million Americans and is thefourth leading cause of death in the US. According to the World HealthOrganization in 2007, 210 million people worldwide have suffered fromCOPD and 3 million of those died in 2005 alone⁶⁸. According to thecurrent trends, WHO estimates that it will become the third leadingcause of death worldwide by the year 2030.

Although a number of different mechanisms may be responsible for theloss of alveoli in pulmonary emphysema, damage to elastic fibers is asignificant factor in the pathogenesis of this disease^(1,2). Thesefibers are responsible for the mechanical recoil that facilitates theexpiration of air from the lungs, and their breakdown can lead toalveolar distention and rupture.

Elastin fibers are part of the extracellular matrix and are an essentialstructural component of lung, skin, and blood vessels. Desmosine (D) andIsodesmosine (I) are two unique pyridinium amino acids that serve ascrosslinking molecules binding the polymeric chains of amino acids intothe 3D network of elastin³³⁻³⁵. The degradation of elastin-containingtissues that occurs in several widely prevalent diseases, such asatherosclerosis, aortic aneurysms, cystic fibrosis and COPD whichincludes pulmonary emphysema etc., have been associated with increasedexcretion in the urine of peptides containing these two pyridiniumcompounds³⁶⁻⁴⁴. As noted above, in lung, elastin degradation occurs withthe development of COPD related to smoking or related to α1-antitrypsindeficiency (AATD)^(45,46).

Due to the importance of elastic fiber injury in pulmonary emphysema, anumber of therapeutic approaches have focused on protecting thisextracellular matrix component from degradation by elastases and otherinjurious agents^(3,4). However, determining the effectiveness of suchtreatment is often difficult because of the lack of sensitive, real-timeindicators of successful therapeutic intervention. Both pulmonaryfunction tests and high-resolution computerized tomography (HRCT)require prolonged time intervals to assess the potential benefits of atherapy, while more sensitive markers such as proinflammatory cytokineslack the necessary specificity to determine efficacy⁵⁻⁹. One possiblesolution to this problem is measurement of elastic fiber breakdownproducts themselves.

Desmosine and isodesmosine, the crosslinking amino acids present only inelastin in the human, offer the prospect of assessing elastindegradation in disease by their measurement in certain body fluids. Thusfar, D and I have been measured in urine of patients with COPD and foundto be statistically significantly elevated above normal controls. Onestudy demonstrated the daily variability of excretion of desmosine andisodesmosine and did not show a statistically significantly elevatedexcretion of these amino acids in patients in 24-hour collections. Inthis same study, statistically significantly increased excretion ofdesmosine and isodesmosine was found in patients with cystic fibrosis.

Various techniques including RIA^(47,48), HPLC⁴⁹⁻⁵¹, and capillary zoneelectrophoresis^(52,53) have been utilized for the analysis of urinary Dand I. Measurements in subjects with COPD show increased levels of D andI in acid hydrolyzed sputum and plasma, along with elevated free D and Iin urine without acid hydrolysis^(11,54).

Recent advances in detecting the elastin-specific amino acid crosslinksdesmosine and isodesmosine have greatly increased the sensitivity andspecificity of this test procedure¹⁰⁻¹⁴. Levels of D and I in urine andplasma have been shown to correlate with physiological and radiologicalmeasures of COPD¹².

Peptides of elastin have been measured in plasma by radioimmunoassay(RIA) and found to be elevated in patients with COPD. Because ofvariability of the specificity of antibodies to elastin peptides in suchRIAs, however, quantitation of such peptides has varied among variousstudies. Advances have been made in the ability to measure D and I incertain complex biological samples using mass spectrometry. For example,the LC/MS analysis which provides increased sensitivity and specificitymay be an important method for biomarker analysis of elastin degradationin disease⁵⁵.

Three LC/MSMS analyses of D and I have been reported⁵⁶⁻⁵⁸. These studieshave been limited to the analysis of D and I content in urine or mouselung hydrolysates. As we have shown in the study of D and I inCOPD^(11,54) and a study on the effect of Tiotropium therapy on D and Ilevels in COPD¹⁰, it is recommended that D and I levels be evaluated inother body fluids as well; such as sputum, plasma, or bronco alveolarlavage fluid. An accurate and reproducible quantification of D and I inseveral body fluids may be more useful to characterize elastindegradation in disease and to follow the course of disease andtherapies⁵⁹. In addition, two recent FDA workshops sponsored by the COPDFoundation held in May, 2009 and January, 2010 have called forstandardization of the analysis to provide practical clinical biomarkersfor COPD.

Active smoking is the most important modifiable risk factor for COPD⁶⁹.In comparison to previous years, overall less people smoke now, however,there has been a disturbing increase in active smokers in the under-30age group⁶⁸. Once active smoking was established as having detrimentalhealth effects on lung disease, the focus turned to the possible adverseeffects of passive smoking—the atmospheric exposure to second handsmoke.

Second Hand Smoke exposure (SHS) has been implicated as a risk factor inmany diseases including asthma, bronchitis, and coronary arterydisease⁷⁰. As a result, there is a worldwide campaign to eliminatepassive smoking from the environment⁷¹. Second hand smoke increases therisk of heart disease in adults⁷² and has been shown to increase theinflammatory state⁷³. Flouris et al. demonstrated an increase ininflammatory cytokines in individuals who had never smoked who wereexposed to only one hour of second hand smoke, and these cytokinesremained elevated for three hours after the exposure ceased⁷⁰. In theAttica study, inflammatory cytokines were elevated from chronic exposureto second hand smoke for extended periods, and the levels were similarto these of the active smokers⁷⁴.

Given the deleterious effects of passive smoking on overall health, itis reasonable to consider its detrimental effects on lung parenchymaitself. Studies of subjects exposed heavily to second hand smoke, i.e.bar workers, flight attendants, have shown an increase in lung cancer,COPD, bronchitis, and asthma exacerbations⁷⁵. In the same studies,subjective health improved within one month of a clean air environmentand objective improvement was seen within two months. There are studiesdemonstrating the effect of second hand smoke exposure on biomarkers ofinflammation in patients with cardiovascular as well as pulmonarydisease⁷⁶⁻⁷⁸. However thus far, no studies have evaluated the effect ofsecond hand smoke exposure on tissue matrix proteins.

In view of the foregoing, there is a need for methods of accuratelydetecting and measuring elastin components, such as desmosine,isodesmosine or combinations thereof, for the purpose of diagnosingand/or treating COPD, chronic bronchitis, emphysema, refractory asthma,and other related diseases and/or monitoring patients with such diseasesand/or who are exposed to, e.g., SHS. Similarly, there is a need formethods of validating whether a candidate compound is effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury.

SUMMARY OF THE INVENTION

We have developed a more specific and sensitive LC/MS analysis, whichcan measure D and I in plasma, urine, and for the first time in sputum.According to one preferred embodiment of the present invention, methodsare provided for validating whether a candidate compound is effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury. In such embodiments, the methods comprisedetermining if the candidate compound decreases the degradation ofelastic fiber in a patient administered the candidate compound bymeasuring, using mass spectrometry, a marker of elastic fiberdegradation in a sample of a body fluid or a tissue of the patient. Theinvention provides that a decrease in the presence of the markercompared to a control validates that the candidate compound is effectiveto treat, prevent, or ameliorate the disease.

According to another preferred embodiment of the present invention,methods are provided for validating whether a candidate compound iseffective to treat, prevent, or ameliorate the effects of COPD. Suchmethods comprise determining if the candidate compound decreases thedegradation of elastin in a patient administered the candidate compoundby measuring, using mass spectrometry, the amount of desmosine,isodesmosine, or combinations thereof in a sample of a body fluid ortissue of the patient. The invention provides that a decrease in thepresence of desmosine and/or isodesmosine compared to a controlvalidates that the candidate compound is effective to treat, prevent, orameliorate the disease. In certain preferred embodiments, the body fluidmay comprise plasma, urine, sputum, or combinations thereof.

According to additional embodiments of the present invention, methodsare provided for identifying candidate compounds that are effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury. Such methods of the invention comprise (a)administering a candidate compound to a cell culture model of thedisease; (b) measuring, by mass spectrometry, the amount of a marker ofelastic fiber injury in the cell culture administered the candidatecompound; and (c) determining whether the amount of the marker producedby the cell culture administered the candidate compound is differentcompared to a control cell culture absent the candidate compound.Non-limiting examples of appropriate markers include desmosine,isodesmosine, or combinations thereof. The invention provides that adecrease in the amount of such marker(s) produced by the cell cultureadministered the candidate compound compared to the control cell cultureidentifies the candidate compound as effective to treat, prevent, orameliorate the effects of the disease.

According to one preferred embodiment of the present invention, methodsare provided for validating whether a candidate compound is effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury. In such embodiments, the methods comprisedetermining if the candidate compound decreases the degradation ofelastic fiber in a patient administered the candidate compound bymeasuring, using mass spectrometry employing an internal standard, amarker of elastic fiber degradation in a sample of a body fluid or atissue of the patient. The invention provides that a decrease in thepresence of the marker compared to a control validates that thecandidate compound is effective to treat, prevent, or ameliorate thedisease. In preferred methods, tandem mass spectrometry is used.

According to another preferred embodiment of the present invention,methods are provided for validating whether a candidate compound iseffective to treat, prevent, or ameliorate the effects of COPD. Suchmethods comprise determining if the candidate compound decreases thedegradation of elastin in a patient administered the candidate compoundby measuring, using mass spectrometry employing an internal standard,the amount of desmosine, isodesmosine, or combinations thereof in asample of a body fluid or tissue of the patient. The invention providesthat a decrease in the presence of desmosine and/or isodesmosinecompared to a control validates that the candidate compound is effectiveto treat, prevent, or ameliorate the disease. In certain preferredembodiments, the body fluid may comprise plasma, urine, sputum, orcombinations thereof. In preferred methods, tandem mass spectrometry isused.

According to additional embodiments of the present invention, methodsare provided for identifying candidate compounds that are effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury. Such methods of the invention comprise (a)administering a candidate compound to a cell culture model of thedisease; (b) measuring, by mass spectrometry employing an internalstandard, the amount of a marker of elastic fiber injury in the cellculture administered the candidate compound; and (c) determining whetherthe amount of the marker produced by the cell culture administered thecandidate compound is different compared to a control cell cultureabsent the candidate compound. Non-limiting examples of appropriatemarkers include desmosine, isodesmosine, or combinations thereof. Theinvention provides that a decrease in the amount of such marker(s)produced by the cell culture administered the candidate compoundcompared to the control cell culture identifies the candidate compoundas effective to treat, prevent, or ameliorate the effects of thedisease. In preferred methods, tandem mass spectrometry is used.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: HPLC separation of D and I was achieved by an Atlantis dC18column (2.1×150 mm, 3 μm) (Waters). The mobile phase A is aqueous 7 mMheptafluorobutyric acid and 5 mM ammonium acetate, and the mobile phaseB is a solution of 7 mM heptafluorobutyric acid and 5 mM ammoniumacetate in a acetonitrile/water (8:2). HPLC chromatography was performedusing a 12 minute linear gradient flow of the mobile phase A from 100%to 88% and mobile phase B from 0% to 12% at a flow rate of 0.3 ml/min.The temperature of the HPLC column is set at 30° C. Under thesechromatographic conditions desmosine and isodesmosine were detected at8.95 minutes and 9.90 minutes, respectively. The mass spectrometer wasoperated in the positive ion mode with the following spectrometricparameters: capillary voltage 3.20 kV, sample cone voltage 55 V, ionenergy 0.5 eV, amplifier voltage 650 V, and temperatures of thedesolvation and the source at 400° C. and 120° C., respectively.

FIG. 1B: Quantification of D and I was achieved by a single ion record(SIR) of D and I molecular ions, both at m/z 526.25 (two isomericmolecules), produced from the LC/MS analysis. Peak areas of the SIRobtained by D and I standards provided good linearity between 0.05 ng to5 ng.

FIG. 2: Shown in FIG. 2 are mean levels and standard errors of the meanof D and I in plasma for normal controls, patients with COPD withoutalpha-1 antitrypsin deficiency (AATD) and patients with COPD with AATD.The differences among all three groups are statistically significant.P-values are calculated based on the summed values of D and I using theunpaired t-test with Welch's correction.

FIG. 3: Shown in FIG. 3 are mean levels and standard errors of the meanof D and I in urine of normal controls, patients with COPD without AATDand COPD with AATD. Mean differences among the groups are statisticallysignificant for the free D and I and % of free/total D and I urinaryexcretion. P-values calculated as in FIG. 1.

FIG. 4: Shown in FIG. 4 are mean levels and standard error of the meanof D and I in sputum of patients with COPD without AATD and COPD withAATD. Control subjects do not have detectable D or I in induced sputum.The content of D and I in sputum of patients with COPD and AATD isstatistically significantly higher than in those with COPD and withoutAATD. P-values calculated as in FIG. 1.

FIG. 5: A table summarizing the experimental results described hereinrelative to COPD patients having normal alpha 1-antitrypsin.

FIG. 6: A table summarizing the experimental results described hereinrelative to patients having alpha 1-antitrypsin related-COPD.

FIG. 7: FIG. 7 demonstrates increasing specificities of three analyticalmethods. The HPLC/UV method measures all molecular species that have thesame UV absorption (286 mμ) with D and I. The SIM method identifies andquantifies all molecules that have the same molecular weight (526) withD and I. The RIM method identifies and quantifies the ion fragments (481and 397) that are only derived from D and I.

FIG. 8: Shown in FIG. 8 are measurement of D and I in plasma usingvarious methods (HPLC/UV (A), SIM (B), and RIM (C)) described in FIG. 7.This figure demonstrates increasing specificity or sensitivity of themethods using a sample of 0.3 ng D/I in 0.5 mL of COPD plasma. The massspectrometric method (LC/MS and LC/MS/MS) for the measurement of D/I inurine, plasma and sputum is more sensitive and specific than existingradioimmunoassays and HPLC methods.

FIG. 9: A table summarizing the experimental results described hereinrelative to patients treated with Tiotropium.

FIG. 10: Shown in FIG. 10 are percent decrease in D/I levels in urine,plasma, and sputum from patients before treatment and two months aftertreatment with Tiotropium.

FIG. 11: Shown in FIG. 11 are peaks of D and I standards in the toppanel, as obtained from LC/MSMS analysis. The peak for acetylatedpyridinoline (IS), is shown in the lower panel. Chromatographic peakareas under the curves of the standards are used in the calculation of Dand I concentrations in samples.

FIG. 12: Shown in FIG. 12 are calibration curves for D, I and IS afterCF-1 treatment. The left panel shows the calibration curves for D and I.The right panel shows the calibration curve for IS.

FIG. 13: Shown in FIG. 13 are the formulas used to determine theconcentration of D and of I in the samples. In the calculations, theratio of the analyte response to the internal standard response is takeninto account.

FIGS. 14A-14D: Shown are spectrograms for samples from a patient ofurine (free D and I) (FIG. 14A), urine (total D and I) (FIG. 14B),plasma (FIG. 14C) and sputum (FIG. 14D), wherein each sample was testedthree times. The left panel of each shows the spectrogram for IS and theright panel of each shows the spectrograms in which D and I are resolvedfrom the sample.

FIG. 15: The levels of BALF DID in both treatment groups showed a markeddecrease after 4 months of smoke exposure. Although differences betweenthe groups were not statistically significant at 2 and 4 months,subsequent measurements in the HA-treated animals were significantlylower (n 3 for each bar). Levels of DID were undetectable (≦1 pg/mgprotein) in 8-week-old animals that were not exposed to either HA orsmoke (not shown; n=4).

FIG. 16: An increase in lung DID was seen in both treatment groupsduring the first 2 months of smoke exposure, followed by a decrease overthe next 4 months and a second increase between 6 and 10 months (n≧3 foreach bar). Differences between the groups were not statisticallysignificant over the 10-month period following initial smoke exposure.

FIG. 17A: Lungs exposed to smoke for 3 months without HA treatmentshowed a moderate degree of airspace enlargement.

FIG. 17B: Lungs exposed to smoke for 3 months and treated with HA for 2months showed only minimal airspace enlargement. Original magnificationof both figures: 40× (hematoxylin and eosin).

FIG. 18: After 3 months of smoke exposure, animals treated with HAshowed a significant reduction in airspace enlargement compared to thosereceiving smoke alone (n=5 for each bar).

FIG. 19A: Photomicrograph of a mouse lung treated with HA and exposed tocigarette smoke for 3 months showing papillary hyperplasia of bronchialepithelium and proliferation of elastic fibers (arrows).

FIG. 19B: Similar histological changes were seen in lungs receivingsmoke alone for the same period of time. Original magnification of bothfigures: 400× (Verhoef-Van Gieson).

FIG. 20A: Molecular structures of Desmosine (DES), Isodesmosine (IDS),and Acetylated Pyridinoline (IS).

FIG. 20B: LC/MSMS chromatogram of DES, IDS, and IS.

FIG. 21A: Linearity response of DES and IDS analysis. Differentconcentrations of DES/IDS (1.25-125 pg) were added with 50.0 pg of IS,absorbed on CF1 cartridge columns, washed and eluted DES/IDS werequantitated separately. FIG. 21B: Same as FIG. 21A, but DES+IDS werequantitated.

FIGS. 22A-22C: DES/IDS levels in COPD patients and healthy controls: Theboundaries of the box indicate between the 25th to 75th percentile, andthe line within the box is the median. FIG. 22A: The level (mg/gcreatinine) of urinary DES+IDS in COPD patients (n=11) and healthycontrols (n=9). (1) free DES+IDS of COPD (median 3.69) versus controls(median 2.39), p=0.0098; (2) total DES+IDS of COPD (median 11.50) versuscontrols (median 9.67), p=0.1147; (3) ratio of free DES+IDS to totalDES+IDS of COPD (median 39.5) versus controls (median 24.3), p=<0.0001.FIG. 22B: The levels of plasma total DES+IDS in COPD (n=14) and healthycontrols (n=4). (1) total DES+IDS (ng/ml) of COPD (median 0.40) versuscontrols (median 0.21), p<0.0001 (2) total DES+IDS (ng/g protein) ofCOPD (median 7.37) versus controls (median 3.76), p=0.0001. FIG. 22C:The levels of sputum of total DES+IDS in COPD (n=8) and healthy controls(n=5). (1) total DES+IDS (ng/ml) of COPD (median 0.22) versus controls(below LOD limit 0.04 ng/ml), p=0.0093; (2) total DES+IDS (ng/g protein)of COPD (median 56.94) versus controls (below LOD 14.29 ng/g protein),p=0.0025.

FIG. 23A: Desmosine/Isodesmosine (D/I) levels in plasma of 98 subjects(Cohort I). The boundaries of the box indicate between the 25^(th) to75^(th) percentile, the line within the box is the median, the whiskersshow the ranges of data points. The D/I levels are illustrated for: a)individuals that have not been exposed to second-hand smoke(“non-exposed”) (n=30), median 0.22 ng/ml; b) individuals that have beenexposed to second-hand smoke (“exposed”) (n=34), median 0.27 ng/ml; andc) smokers (“smokers”) (n=34), median 0.31 ng/ml. The P values for thefollowing comparisons are also provided: non-exposed vs. exposed,P=0.0050; exposed vs. smokers, p=0.0533; non-exposed vs. smokers,p<0.0001.

FIG. 23B: Cotinine levels in plasma of 98 subjects (Cohort I). Theboundaries of the box indicate between the 25^(th) to 75^(th)percentile, the line within the box is the median, the whiskers show theranges of data points. The cotinine levels are illustrated for: a)individuals that have not been exposed to second-hand smoke(“non-exposed”) (n=30), median 0 ng/ml; b) individuals that have beenexposed to second-hand smoke (“exposed”) (n=34), median 0 ng/ml; and c)smokers (“smokers”) (n=34), median 9.36 ng/ml. The P values for thefollowing comparisons are also provided: non-exposed vs. exposed,P=0.1161; exposed vs. smokers, p=0.2117; non-exposed vs. smokers,p=0.0012

FIG. 24A: Desmosine/Isodesmosine (D/I) levels in plasma of 22 subjects(Cohort II). The boundaries of the box indicate between the 25^(th) to75^(th) percentile, the line within the box is the median, the whiskersshow the ranges of data points. The D/I levels are illustrated for: a)individuals that have not been exposed to second-hand smoke(“non-exposed”) (n=7), median 0.21 ng/ml; b) individuals that have beenexposed to second-hand smoke (“exposed”) (n=6), median 0.40 ng/ml; andc) smokers (“smokers”) (n=9), median 0.45 ng/ml. The P values for thefollowing comparisons are also provided: non-exposed vs. exposed,P=0.0223; exposed vs. smokers, p=0.1782; non-exposed vs. smokers,p=0.0075.

FIG. 24B: Cotinine levels in plasma of 22 subjects (Cohort II). Theboundaries of the box indicate between the 25^(th) to 75^(th)percentile, the line within the box is the median, the whiskers show theranges of data points. The cotinine levels are illustrated for: a)individuals that have not been exposed to second-hand smoke(“non-exposed”) (n=6), median 0.84 ng/ml; b) individuals that have beenexposed to second-hand smoke (“exposed”) (n=5), median 0.80 ng/ml; andc) smokers (“smokers”) (n=9), median 11.8 ng/ml. The P values for thefollowing comparisons are also provided: non-exposed vs. exposed,P=0.4008; exposed vs. smokers, p=0.0740; non-exposed vs. smokers,p=0.3470.

FIG. 25: Cohort I: A statistically significant correlation is shownbetween the log values of plasma cotinine concentration and plasmalevels of D/I in cohort 1.

FIG. 26: HPLC/MS base peak chromatograms of human lung EDP's (<10,000Da) using (A) HNE digestion; and (B) MMP12 digestion.

FIG. 27: Annotated MS/MS spectrum of peptide VGVLPGVPT (SEQ ID NO:16)(m/z 838) with alignment by de novo PEAK assignment of the amino acidsequence.

FIG. 28: SRM LC/MS spectra of patient 1's plasma sample (top panels) and10 ng/ml standard peptides (bottom panels). Transition ions: (A) 449.24to 229.05; (B) 499.32 to 297.09; (C) 632.35 to 446.21; (D) 838.48 to622.49 were used for identification and quantitation of the 4 peptides.

FIG. 29: Ion chromatograms of hexapeptides (m/z 499) derived fromhydrophobic elastomeric repeats. Ion chromatograms are illustrated foreach of the following sample digestions (a) digestion of lung elastin byHNE; (b) digestion of lung elastin by MMP12; (c) digestion of lungelastin by PPE; (d) digestion of ligament elastin by HNE; (e) digestionof ligament elastin by PPE.

DETAILED DESCRIPTION OF THE INVENTION

According to one preferred embodiment of the present invention, methodsare provided for validating whether a candidate compound is effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury, such as elastin degradation. In such embodiments,the methods comprise determining if the candidate compound decreases thedegradation of elastic fiber in a patient administered the candidatecompound by measuring, using mass spectrometry, a marker of elasticfiber degradation in a sample of a body fluid or a tissue of thepatient. The invention provides that a decrease in the presence of themarker compared to a control validates that the candidate compound iseffective to treat, prevent, or ameliorate the disease.

The foregoing methods may be used to validate whether a candidatecompound is effective to treat, prevent, or ameliorate the effects ofchronic obstructive pulmonary disease (COPD), chronic bronchitis,emphysema, and/or refractory asthma. The marker of elastic fiberdegradation that is measured using mass spectrometry is preferablydesmosine, isodesmosine, or combinations thereof. In such embodiments,the marker(s), such as desmosine, isodesmosine, or combinations thereof,are preferably detected and measured within a patient's urine, plasma,and/or sputum.

In certain preferred embodiments of the invention, desmosine,isodesmosine, or combinations thereof are measured in plasma. In certainalternative embodiments, total free desmosine, isodesmosine, orcombinations thereof are measured in urine. The methods of the presentinvention may be employed to test the therapeutic value, oreffectiveness, of a variety of different candidate compounds.Non-limiting examples of such compounds include hyaluronic acid,polysaccharides, carbohydrates, small molecules, and RNAi molecules,including siRNAs, shRNAs, and others.

According to additional embodiments of the present invention, methodsare provided for identifying candidate compounds that are effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury. Such methods of the invention comprise (a)administering a candidate compound to an in vivo or in vitro model ofthe disease, e.g., a cell culture; (b) measuring, by mass spectrometry,the amount of a marker of elastic fiber injury in the cell cultureadministered the candidate compound; and (c) determining whether theamount of the marker produced by e.g., the cell culture administered thecandidate compound is different compared to e.g., a control cell cultureabsent the candidate compound. Non-limiting examples of appropriatemarkers include desmosine, isodesmosine, or combinations thereof. Theinvention provides that a decrease in the amount of such marker(s)produced by e.g., the cell culture administered the candidate compoundcompared to e.g., the control cell culture identifies the candidatecompound as effective to treat, prevent, or ameliorate the effects ofthe disease.

Such methods may be used for identifying candidate compounds that areeffective to treat, prevent, modulate and/or ameliorate the effects ofelastin degradation and diseases associated therewith, such as COPD,chronic bronchitis, emphysema, and/or refractory asthma. Similar to theother embodiments discussed herein, the marker that is measured by massspectrometry is preferably selected from desmosine, isodesmosine, orcombinations thereof. Still further, similar to the other embodimentsdiscussed herein, such methods may be employed to test the therapeuticvalue, or effectiveness, of a variety of different candidate compounds,including hyaluronic acid, polysaccharides, carbohydrates, smallmolecules, and RNAi molecules, such as siRNAs, shRNAs, and others.

It is noted that, throughout the instant disclosure, desmosine isfrequently abbreviated as “D” or “DES,” and isodesmosine is frequentlyabbreviated as “I” or “IDS.” Similarly, desmosine and isodesmosine maybe collectively abbreviated herein as “D and I,” “D/I,” “DID,”“DES/IDS,” or “DES and IDS.”

“Tandem mass spectrometry” as used herein refers to techniques in whicha sample is analyzed two or more times by mass spectrometry. Typically,the sample is analyzed twice, which is referred to herein as MS/MS orMSMS, but it may be analyzed three or more times. In tandem massspectrometry, the same mass spectroscope may be used two or more timesfor a given sample, or separate mass spectroscopes may be used. In thelatter case, preferably two different mass spectroscopes connected inseries are used. The first mass spectrometer sorts and weighs thesample, then the sample enters a collision cell which breaks the sampleinto fragments, and the second mass spectrometer sorts and weighs theresulting fragments.

Preferably, the technique used to analyze D and I in a sample isLC-MS/MS. This is shown, for example, in FIG. 7. Following HPLCseparation, the chromatographic peaks are analyzed by tandem massspectrometry and measured by selected reaction monitoring (SRM) of twoions m/z 481 and m/z 397, which are two distinct fragment ions producedby collision-induced dissociation (CID) of the molecular ion (m/z 526)of D/I. Q1, Q2, and Q3 in FIG. 7 represent three quadropoles in which Q1and Q3 are mass sorters and Q2 is used as a collision cell.

More preferably, an internal standard is used which is included witheach sample tested. An internal standard that bears a close structuralsimilarity to D and I, an acylated pyridinoline, more preferablyacetylated pyridinoline (IS), was used. The structural similarity can beseen in a comparison of the structures, as follows:

According to one preferred embodiment of the present invention, methodsare provided for validating whether a candidate compound is effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury, such as elastin degradation. In such embodiments,the methods comprise determining if the candidate compound decreases thedegradation of elastic fiber in a patient administered the candidatecompound by measuring, using mass spectrometry employing an internalstandard, a marker of elastic fiber degradation in a sample of a bodyfluid or a tissue of the patient. The invention provides that a decreasein the presence of the marker compared to a control validates that thecandidate compound is effective to treat, prevent, or ameliorate thedisease. In preferred methods, tandem mass spectrometry is used.

The foregoing methods may be used to validate whether a candidatecompound is effective to treat, prevent, or ameliorate the effects ofchronic obstructive pulmonary disease (COPD), chronic bronchitis,emphysema, and/or refractory asthma. The marker of elastic fiberdegradation that is measured using mass spectrometry employing aninternal standard is preferably desmosine, isodesmosine, or combinationsthereof. In such embodiments, the marker(s), such as desmosine,isodesmosine, or combinations thereof, are preferably detected andmeasured within a patient's urine, plasma, and/or sputum. In preferredmethods, tandem mass spectrometry is used.

In certain preferred embodiments of the invention, desmosine,isodesmosine, or combinations thereof are measured in plasma. In certainalternative embodiments, total free desmosine, isodesmosine, orcombinations thereof are measured in urine. The methods of the presentinvention may be employed to test the therapeutic value, oreffectiveness, of a variety of different candidate compounds.Non-limiting examples of such compounds include hyaluronic acid,polysaccharides, carbohydrates, small molecules, and RNAi molecules,including siRNAs, shRNAs, and others.

According to additional embodiments of the present invention, methodsare provided for identifying candidate compounds that are effective totreat, prevent, or ameliorate the effects of a disease characterized byelastic fiber injury. Such methods of the invention comprise (a)administering a candidate compound to an in vivo or in vitro model ofthe disease, e.g., a cell culture; (b) measuring, by mass spectrometryemploying an internal standard, the amount of a marker of elastic fiberinjury in the cell culture administered the candidate compound; and (c)determining whether the amount of the marker produced by e.g., the cellculture administered the candidate compound is different compared toe.g., a control cell culture absent the candidate compound. Non-limitingexamples of appropriate markers include desmosine, isodesmosine, orcombinations thereof. The invention provides that a decrease in theamount of such marker(s) produced by e.g., the cell culture administeredthe candidate compound compared to e.g., the control cell cultureidentifies the candidate compound as effective to treat, prevent, orameliorate the effects of the disease. In preferred methods, tandem massspectrometry is used.

Such methods may be used for identifying candidate compounds that areeffective to treat, prevent, modulate and/or ameliorate the effects ofelastin degradation and diseases associated therewith, such as COPD,chronic bronchitis, emphysema, and/or refractory asthma. Similar to theother embodiments discussed herein, the marker that is measured by massspectrometry employing an internal standard is preferably selected fromdesmosine, isodesmosine, or combinations thereof. Still further, similarto the other embodiments discussed herein, such methods may be employedto test the therapeutic value, or effectiveness, of a variety ofdifferent candidate compounds, including hyaluronic acid,polysaccharides, carbohydrates, small molecules, and RNAi molecules,such as siRNAs, shRNAs, and others. In preferred methods, tandem massspectrometry is used.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

Example 1—D and I Measurements

In these examples, measurements of desmosine (D) and isodesmosine (I) inplasma, urine and sputum are described. The results demonstrate astatistically significant difference between normal controls andpatients diagnosed with COPD and further suggest that measurements of Dand I in plasma may be a discriminating index for distinguishingpatients with COPD from normal subjects. D and I were measured inplasma, urine and sputum in a cohort of patients diagnosed with COPDrelated to smoking and a second cohort in whom COPD is related toZ-phenotype alpha-1 antitrypsin deficiency (AATD) as well as smoking.

Materials and Methods

The mass spectrometric method was used for direct measurement of D/I inurine, plasma and sputum as markers of elastin degradation in healthycontrols, patients with α1-antitrypsin deficiency (AATD) andnon-AATD-related COPD. Preparation of specimens of urine and sputum andmeasurements by mass spectrometry (LC/MS) were performed as previouslydescribed in Ma S, Lieberman S, Turino G M and Lin Y Y: The detectionand quantitation of free desmosine and isodesmosine in human urine andtheir peptide-bound forms in sputum¹¹. D and I standard (mixed 50% D and50% I) were purchased from Elastin Products (Owensville, Mich.), and allother reagents were from Sigma (St. Louis, Mo.). MCX cation exchangecartridges (3 ml) were obtained from Waters (Milford, Mass.), and CF1cellulose powders were purchased from Whatman (Clifton, N.J.).

Urine Samples.

Twenty-four hour urine samples were collected and analyzed as previouslydescribed¹¹.

Plasma Samples.

Plasma samples were obtained after centrifuging venous blood specimensat 2500 r.p.m. for 25 min. Samples were stored at −20° C. until used.One ml of plasma and 1 ml of concentrated HCl (37%) were placed in aglass vial. After air in the sample was displaced with a stream ofnitrogen, the sample was acid hydrolyzed for 24 hours in 6N HCl. Afterevaporation to dryness, the residue was dissolved in 2 ml of a mixedsolution of n-butanol/acetic acid/6 N HCl (4:1:1, by volume). The samplesolution was loaded onto a 3 ml CF1 cartridge. The CF1 cartridge wasprepared by introducing 3 ml of the slurry of 5% CF1 cellulose powder ina mixture of n-butanol/acetic acid/water (4:1:1, by volume). Thecartridge was washed 3 times with 3 ml of n-butanol/acetic acid/watermixture, and D and I adsorbed in the CF1 cartridge were eluted with 3 mlof water. The eluate was evaporated to dryness under vacuum at 45° C.and the residue was dissolved in 0.1 ml of HPLC mobile phase for LC/MSanalysis. For analysis in plasma, samples were processed and measured induplicate and the results averaged.

Sputum Samples.

Sputum samples were processed as previously described¹¹ with thefollowing modification: The acid hydrolyzed samples were chromatographedusing a CF1 cartridge as described in the treatment of plasma samples.Each sputum sample was processed and measured in duplicate and theresults averaged. Sputum was obtained from 3-hour morning collectionsspontaneously produced. When subjects could not voluntarily producesputum, sputum induction was induced by 3% saline inhalation for 20minutes as previously described¹¹.

Recovery of Desmosine and Isodesmosine in Urine and Plasma.

Using D and I as the external standards we performed studies to ensurerecovery and reproducibility of the analysis in urine and plasma.Triplicates of two urine samples, were spiked with 0.4 pmol and 2.0 pmoleach of D and I standards, and carried through HCl hydrolysis andchromatography procedures as described. The recoveries of D and I fromone urine spiked with 2.0 pmol of D and I were 91±4% and 88±1%, and thatspiked with 0.4 pmol of D and I were 92±3% and 93±8%. The recoveries ofD and I from the other urine spiked with 2.0 pmol of D and I were 88±1%and 93±3%, and that spiked with 0.4 pmol of D and I were 93±6% and93±15%. The reproducibility of the repeated sample analysis ranges from91-99%.

Similar recovery studies were carried out with 4 plasma samples. Therecoveries of D and I with 0.05 ng standards were 65±4 and 74±13%, andthat with 0.1 ng standards were 67±1 and 72±4%. The reproducibility ofthe repeated sample analysis is 83-99%. Values in urine and plasma werecorrected for recovery losses.

Creatinine and Protein Measurement were carried out as previouslydescribed¹¹. LC/MS Analysis was performed also as previouslydescribed¹¹, with slight modification (see description of FIG. 1A).

Data Analysis.

The t-test adjusted for unequal variance was used to test the nullhypothesis. The level of significance was 0.05. P-values were calculatedbased on the summed values of D and I using the unpaired t-test withWelch's correction.

Patients.

Study patients were diagnosed with chronic obstructive pulmonary diseaseand adhere to Gold Criteria grades 1-4. All patients were screened forAATD by serum levels and phenotyping. Patients were divided into twogroups: 1) with normal levels of alpha-1 antitrypsin in serum, and 2)those with ZZ-homozygous alpha-1 antitrypsin deficiency. Patients gaveinformed consent for the study. The study was approved by theInstitutional IRB.

All patients with normal levels of alpha-1 antitrypsin had significantsmoking histories of from 10 to 60 pack years. Many had stopped in theprevious ten years and none were current smokers when studied. Amongthese patients the age range was 44 to 85. Five were males and 2females.

Among patients with alpha-1 antitrypsin deficiency all but one had asignificant smoking history exceeding ten pack years. All patients hadceased smoking for at least ten years by the time of study. All AATDpatients were being treated with AAT protein replacement, were in astable clinical state and exhibited no evidence of an exacerbation.

Control subjects were selected by a clinical history free of anyspecific known disease or significant symptoms, including respiratorysymptoms, and none had ever smoked.

Results

Results in normal subjects are presented in Table 1 below (C=Caucasian;A=Asian).

TABLE 1 Controls without Lung Disease Desmosine/Isodesmosine UrinePlasma Free Form Free/ ng/g μg/g Total Subjects Sex Age Race ng/mlprotein creatinine Total % 1 M 33 C 0.11/0.10 1.89/1.80 1.85/1.119.64/5.90 19/19 2 M 35 C 0.07/0.09 1.06/1.36 3 F 58 C 0.10/0.082.17/1.74 3.73/2.76 10.22/7.65  36/36 4 M 27 A 0.09/0.07 1.31/1.020.60/0.50 2.85/2.70 21/19 5 F 31 A 0.10/0.06 2.22/1.29 6 F 69 C0.09/0.08 1.62/1.44 1.80/1.69 11.77/8.37  15/20 7 M 54 A 0.11/0.132.02/2.27 0.51/0.64 5.17/3.96 10/16 8 M 72 A 0.09/0.13 1.94/2.800.75/0.35 5.16/4.10 15/9  9 M 79 C 0.12/0.05 2.43/1.01 0.42/0.386.17/4.67 7/8 10 M 65 A 0.11/0.10 2.23/2.03 0.99/0.66 6.59/3.89 15/17 11F 38 A 0.13/0.08 2.27/1.31 0.89/0.88 5.19/4.26 17/21 12 F 28 C 0.11/0.091.83/1.50 2.48/1.58 12.69/6.64  20/24 13 M 32 C 0.10/0.08 1.87/1.491.59/1.56 7.29/5.68 22/27 mean 0.10/0.09 1.91/1.62 1.42/1.10 7.52/5.2618/20 ±SEM ±0.01/±0.01 ±0.11/±0.14 ±0.31/±0.22 ±0.94/±0.53 ±2/±2

The mean levels and standard error (S.E.M.) of D and I (D/I) in plasmain 13 subjects were 0.10±0.01/0.09±0.01 ng/ml plasma and1.91±0.11/1.62±0.14 ng/g protein.

Results for levels of D and I (D/I) in plasma in patients with COPD withnormal levels of AAT are presented in FIGS. 2 and 5. The mean and S.E.M.were 0.39±0.07/0.26±0.07 ng/ml of plasma and 6.60±0.84/4.36±1.04 ng/gprotein, which are statistically significantly higher than controls.Results for levels of D and I in plasma in patients with COPD related toAATD are shown in FIGS. 2 and 6. The mean and S.E.M. are0.78±0.19/0.62±0.14 ng/ml of plasma and 19.24±5.22/15.03±3.71 ng/gprotein, which values are statistically significantly higher thancontrol values and the levels in COPD not related to AATD whencalculated per gm of protein in plasma.

It is noteworthy that no overlap of levels of plasma D and I existsbetween controls and the patient groups with COPD; patients' levels areconsistently higher. The levels of D and I in urine in control subjectsand patients with and without AATD are shown in Table 1 and FIGS. 3, 5and 6. The levels of free D and I (D/I) are 3.66±0.26/2.72±0.21 ng/gcreatinine in COPD with normal levels of AAT and 2.97±0.30/2.15±0.29 inpatients with AATD which values are statistically significantly higherthan control subjects (1.42±0.31/1.10±0.22). As shown in FIG. 3, thepercentage of free D and I over total D and I excretion wasstatistically significantly higher in both groups with COPD, but highestin COPD with normal AAT levels. The mean total 24 hour excretion of Dand I was not statistically significantly increased in both COPD groupsas compared to controls.

Levels of D and I in sputum are shown in FIGS. 4-6. The levels of D andI were below the level of detection by mass spectrometry in 3 controlsubjects, whereas both groups of COPD patients showed mean levels of Dand I to be significantly increased to 1.08±0.26/0.74±0.15 ng/ml and0.30±0.10/0.25±0.09 ng/ml of sputum in COPD patients with and withoutAATD respectively. Results expressed per g of protein in sputum for Dand I (D/I) were 312±115/212±77.9 and 49.9±33.4/43.9±31.5 in patientswith and without AATD. D and I in sputum was statistically significantlyhigher in AATD patients.

Shown in Table 2 below are repeat measurements of plasma D and I in 1control subject, 1 patient with AATD related COPD and a patient withCOPD without AATD. Intervals between repeat measurements were days insubjects with AATD and COPD to weeks and months for the other twosubjects. During these intervals, each patient was in a stable clinicalstate without exacerbations.

TABLE 2 Repeat Measurements of Desmosine and Isodesmosine in Plasma D/I(ng/ml) D/I (ng/g protein) Normal Subject - 14 month interval 0.12/0.052.43/1.01 0.11/0.07 2.11/1.34 Patient with COPD and AATD - 2 dayinterval 2.31/1.75 54.91/41.60 2.53/2.08 55.90/45.96 2.55/2.4961.31/59.87 ng/g protein Patient with COPD without AATD - 6 monthinterval 0.49/0.44 9.32/8.37 0.32/0.31 7.04/6.82

The results varied between 10 and 15%, which suggests a stable metabolicstate with respect to elastin turnover in each individual's normal orabnormal levels.

Levels of D and I (D/I) in plasma and urine were analyzed for possiblecorrelation with age, sex, racial origin or physiological parameters ofFEV₁ and RV/TLC and no statistically significant correlations weredetermined.

Data Analysis

An early insight into the mechanisms leading to alveolar disruption inpulmonary emphysema is that lung matrix elastin is a target for chemicaldegradation from cellular elastases. Lung elastin content, determinedchemically, has been demonstrated to be low in pulmonary emphysemarelated to smoking or to the Z-phenotype AATD, and morphologically, lungelastin fibers have been shown to be fragmented and disordered. Alsointratracheal administration of elastases has uniquely produced animalmodels of pulmonary emphysema. In addition, elastin peptides have beenshown to be chemotactic for neutrophils and macrophages and could be afactor in the progression of human pulmonary emphysema once elastindegradation has occurred.

Current methods of measuring elastin peptides in blood plasma requireradioimmunoassay techniques which depend on antibodies to elastinpeptides which vary in specificity and sensitivity, which affects thestandardization and quantification of peptides. Also, measurements of Dand I in urine require a relatively extensive chemical procedure usingisotope dilution corrections and HPLC, which can be an arduousmethodology.

Recognizing these limitations, mass spectrometry, with its ability todetect specific molecular species with high sensitivity, accuracy andspecificity is a readily applicable method for use in complex bodyfluids. The increased sensitivity of mass spectrometry has permitted themeasurement of a free component unbound to protein or other matrixconstituents of D and I in urine which are increased statisticallysignificantly in patients with COPD as compared with normals. Similarly,mass spectrometry has allowed measurements of D and I in blood plasmaand sputum, both chemically complex media. Attempts to detect a free vs.bound component of D and I in plasma were unsuccessful. Theconcentration of D and I in a single small sample of plasma may be toolow for detection compared to the concentration of D and I in a 24-hourcollection of urine.

The increased free component of D and I in urine in COPD patients, webelieve, may reflect an increased neutrophil elastase concentration incirculating neutrophils, which has been demonstrated by previousmeasurements as an increase in lysosomal elastase in neutrophils of COPDpatients as compared with normals. This increased elastase concentrationmay reflect a generalized immunological hyperreactivity resulting fromthe chronic inflammatory state of the lung in COPD, manifested byincreased elastase activity in neutrophils and macrophages.

The difference in levels of D and I in plasma between controls andpatients with COPD in this study suggests that D and I in plasma may beone of the sensitive indicators of the presence of lung elastinbreakdown in COPD, especially since the entire cardiac output constantlycirculates through the lung. While changes in levels of D and I inplasma cannot be assumed to reflect D and I from lung parenchyma per se,the demonstrated presence of D and I in sputum of patients with COPDindicates that increased degradation, and probably turnover, of elastinis occurring in lung, since normal subjects do not have detectableamounts of D and I in induced sputum.

In the limited number of our controls we did not find any correlation ofthe age of the subjects with urinary excretion or plasma levels of D andI. In other studies of adult subjects which include similar measurementsno correlations with age have been reported.

Measurements of total excretion of D and I in 24 hour urine collectiondid not demonstrate statistically significant differences betweenpatients and normals. This result is consistent with the demonstrationof Bode et al., who showed marked variability in daily excretion of Dand I in COPD patients and no statistically significant difference inthe total excretion between the two cohorts⁴². Also, Starcher et al.have demonstrated a failure of urine to reflect the rapid degradation oflung elastin produced by intratracheal porcine pancreatic elastase inmice. Their studies demonstrated a sequestering of elastin peptides inrenal parenchyma following lung elastin breakdown and a continued slowurinary excretion of D containing peptides over several days followingacute elastase injury¹²². Other studies have shown significant increasesof urinary D in COPD patients compared to normals. Possibly theindividual patient population in the present study varied from thosepreviously studied. In that regard, none of the patients in this studywere actively smoking, which has been shown to increase urinarydesmosine excretion.

When elastin degradation is mildly, or even moderately, increased abovethe turnover in normals, it may be difficult to reflect this increase inurine, even with 24-hour collections. However, the percentage of thefree component of D and I in urine is consistently elevated in bothgroups of patients with COPD.

It has long been demonstrated that elastin in elastin fibers, onceformed, cross-linked and insoluble, is extremely stable and undergoeslittle metabolic turnover. This slow metabolic turnover in normal humansis consistent with the very low levels of D and I in normal plasma. Itis noteworthy that studies of elastase injury to lung elastin in vivo inrats and mice demonstrate that rapid degradation of elastin occurs whenexposed to elastases, with rapidly ascending concentrations of elastinpeptides in blood and urine within hours of protease administration.Notable also is the rapid resynthesis of elastin after proteolyticbreakdown. The stability of plasma and urine levels of desmosine withrepeat measurements over a 44 day interval in patients with AATD wasreported by Stolk et al., which is consistent with measurements in thisstudy¹²³. Thus any increase in elastase activity in lungs, whichincludes bronchial and blood vessel elastin as well as alveolar, maywell be reflected in the circulating blood to and from the lung.

The persistence of elevated levels of D and I in plasma in patients withCOPD in both patient cohorts long after smoking cessation is consistentwith continued inflammation of the lung in COPD and progression ofmatrix tissue injury.

The blood levels of D and I in COPD patients may therefore prove to be asensitive index of the metabolic state of elastin degradation andpossibly resynthesis in the lung. Since elastin is a significantstructural constituent of alveoli, bronchial walls and blood vessels,the levels of D and I in the earliest phases of COPD deserve to beevaluated. Also the responses to therapeutic agents which may reduce thelung inflammatory state and thereby reduce elastin degradation may beassessed by measurements of D and I in plasma and the proportion of freeD and I in urine.

It is noteworthy that the AATD patients had higher levels of D and I inplasma than COPD patients without AATD, along with higher levels insputum consistent with the AATD patients' form of COPD to beemphysematous with loss of lung mass. All patients with AATD werereceiving AAT augmentation therapy at the time of study. Since levels ofD and I in body fluids were not obtained prior to the initiation ofaugmentation therapy, it cannot be assumed that AAT replacement ishaving no beneficial effect. These data suggest that an evaluation ofthe effect on D and I levels of higher doses of AAT augmentation wouldbe worthwhile.

Mass spectrometry allows measurements of D and I separately. Theproportion of D and I in plasma and urine in control subjects shows aslightly lower proportion of isodesmosine constituting approximately 80%of the level of desmosine. In one study of the amino acid composition ofhuman lung elastin, D exceeded I content by approximately 10-15%, whichis close to agreement with the present study¹²⁴. It is noteworthy thatpatients with COPD in both groups had proportions of D and I which aresimilar to controls, suggesting that resynthesis of elastin in thesegroups does not show major structural dissimilarities from normals.

The results of this study indicate that levels of D and I in urine whichincludes an unconjugated fraction, along with levels in plasma andsputum may be useful parameters to characterize patients with COPD ofvarious phenotypes at various phases of the disease. Mass spectrometry,with its increased specificity and sensitivity, should facilitate thischaracterization.

Example 2—Effect of Tiotropium Treatment

COPD patients have elevated levels of D/I in plasma, urine and sputum,which might respond to prolonged bronchodilation. To determine ifclinical effects of Tiotropium (TIO) affect tissue degradation of thelung in COPD, clinically stable patients with COPD (n=9) not on TIOprior to the study and at one month and a second month after initiatingtherapy were tested. Other anticholinergic bronchodilators were stoppedprior to TIO, and other therapies/disease treatments were unchanged forthe two months of study. To these patients, 18 mcg TIO was administeredeach 24 hours. D/I in plasma, urine and sputum were measured by liquidchromatography and mass spectrometry (LC/MS) prior to the study and atone month and two months after the study.

Prior to the study, levels of D/I in plasma and sputum were above normalin all patients studied, and the percentage of free D/I in urine wasalso increased. Significant decreases in D/I levels were observed inurine (10 out of 12), in plasma (10 out of 12) and in sputum (all 12patients), which may reflect decreases in lung elastin degradation ofCOPD patients on TIO therapy. (FIG. 9). Calculated percentage decreasesin D/I levels after TIO treatment showed decreases beginning after onemonth with further decreases observed in the second month. After twomonths of treatment, larger decreases in D/I levels were observed insputum and plasma than urine. The response was not always uniform in therespective patients' urine, plasma, and sputum. For example, twopatients (#3 and #5) failed to show responses in urine but showeddecreases in their plasma and sputum, and two other patients (#1 and #6)did not show decreases in plasma but showed decreases in urine andsputum. (FIG. 10).

Overall results of percent decreases in D/I levels indicated that all 12COPD patients were responding to prolonged TIO treatment with somedecrease in lung elastin degradation. Spirometry in most post-TIOtherapy patients shows significant increase in Force Vital Capacity(FVC), Forced Expired Volume in 1 second (FEV1), and ratio of FEV1/FVCand decreases in Residual Volume (RV). The improvement in spirometricindices were usually concordant with levels of D/I in patients.

Overall results demonstrate that two months of treatment with TIO inpatients is accompanied by significant reductions in D/I levels inplasma, urine and sputum, consistent with a reduction in elastindegradation and possibly an anti-inflammatory effect. Thus, this exampleconfirms the effectiveness of the methods disclosed and claimed hereinfor, e.g., validating whether a candidate compound is effective totreat, prevent or ameliorate the effects of a disease characterized byelastic fiber injury, such as COPD, COPD with AATD, chronic bronchitis,emphysema, or refactory asthma.

Example 3—Analysis Using LC/MSMS and Internal Standard (IS)

D and I concentrations were determined in urine, plasma, and sputumsamples using LC/MSMS with an internal standard which was acetylatedpyridinoline (IS).

Preparation of specimens of urine, plasma, and sputum was performed asdescribed in Example 1. MCX cation exchange cartridges (3 ml) and CF1cellulose powders were as indicated in Example 1. D and I standards(mixed 50% D and 50% I) were obtained from Elastin Products (Owensville,Mich.). Acetylated pyridinoline was obtained from Quidel (San Diego,Calif.). All other reagents were from Sigma (St. Louis, Mo.).

Acid Hydrolysis:

0.2 mL of urine, or 0.5 mL of plasma or sputum were placed in a glassvial with equal volumes of concentrated HCl (37%). Air in the vial wasdisplaced with nitrogen, and was heated at 110° C. for 24 hrs. Thehydrolyzed sample was filtered and evaporated to dryness. For the freeforms of D and I analysis, samples were analyzed directly without theHCl hydrolysis.

Cellulose (CF1) Cartridge Treatment:

The internal standard, acetylated pyridinoline (IS) was added to theacid hydrolyzed and unhydrolyzed (for free D and I) samples; 1 ng wasadded to urine samples and 0.5 ng was added to plasma and sputumsamples. The sample mixtures were dissolved in 2 mL of n-butanol/aceticacid/water (4:1:1), and applied onto a 3 mL cellulose cartridge, whichwas prepared by introduction of 3 mL of 5% CF1 slurry inn-butanol/acetic acid/water (4:1:1) (well dispersed slurry by stirringfor 24 hrs). The cartridge was washed 3 times with 3 mL ofn-butanol/acetic acid/water (4:1:1), and the components retained in thecartridge were eluted with 3 mL of water, dried and dissolved in 200 μlof LC mobile phase.

LC/MSMS Analysis.

A TSQ Discovery electrospray tandem mass spectrometer (Thermoelectron)was used for the LC/MSMS analysis. HPLC separation of D and I wasachieved using a 2 mm×150 mm dC18 (3 μm) column (Waters, Milford, Mass.)with mobile phase A (7 mM HFBA/5 mM NH4Ac in water) and mobile phase B(7 mM HFBA/5 mM NH4Ac in 80% acetonitrile). The HPLC was programmedlinearly from 100% A to 82% A in 12 mins. The tandem mass spectrometry(LC/MSMS) technique monitors ions of m/z 481 and m/z 387. Selectedreaction monitoring (SRM) of D and I (m/z 526 to m/z 481+397) and IS(m/z 471 to m/z 128) were used for the quantitative measurement todetermine D and I concentration in the samples.

Sample Spectrograms.

Spectrograms from analysis of samples from a patient of urine (free Dand I), urine (total D and I), plasma and sputum, in which each samplewas tested three times, are provided in FIGS. 14A-14D.

Calculation.

To determine the concentrations of D and I in the samples, the ratio ofthe analyte response to the internal standard response is ascertained.The calculation used is shown in FIG. 13.

Reproducibility.

From the three measurements taken per sample using LC/MSMS with IS, thecoefficient of variance was calculated.

Urine Urine Free D/I Total D/I (μg/g Creatinine) (μg/g Creatinine) Mean7.45 Mean 15.98 ±SD 0.91 ±SD 1.76 % CV 12 % CV 11

Plasma Sputum D/I (ng/ml) D/I (ng/ml) Mean 0.23 Mean 0.23 ±SD 0.03 ±SD0.01 % CV 13 % CV 4

In comparison, representative samples in which three measurements weretaken per sample were identified from data in which D and I weremeasured using LC/MS. The comparative data is provided as follows:

Urine Urine Free D/I Total D/I (μg/g Creatinine) (μg/g Creatinine) Mean0.93 Mean 9.75 ±SD 0.20 ±SD 0.96 % CV 22 % CV 10

Plasma Sputum D/I (ng/ml) D/I (ng/ml) Mean 0.63 Mean 0.52 ±SD 0.21 ±SD0.11 % CV 33 % CV 21

As can be seen, the coefficient of variance from a representative sampleof urine (Total D/I) measured by LC/MS was about 10%. The otherrepresentative samples, however, had about or above 20% variance. The CV% for an urine (free D/I) sample was 22% and the CV % for a sputumsample was 21%. For a plasma sample, the CV % was as high as 33%.

For the measurements taken using LC/MSMS with IS, on the other hand, thereproducibility was significantly and unexpectedly improved. All of thesamples had a CV % below 15%, and the particular CV percentages spannedfrom 4% (for sputum sample) to 12% (for urine (free D/I) sample). Theuse of tandem MS employing an internal standard shows significantimprovement in reproducibility. Thus, the technique advances theimportance of desmosine and isodesmosine as reliable biomarkers inbiological fluids for the detection of elastin degradation in diseasescharacterized by elastic fiber injury such as COPD. Moreover, using theprocess according to the present invention it is now possible to obtainCV percentages within FDA approved limits (i.e., generally below 15%).

Example 4—Effect of Hyaluronan on Smoke-Induced Elastic Fiber Injury

In the current study, we used the D/I biomarker to determine both theprogression of elastic fiber damage in a mouse model of smoke-inducedpulmonary emphysema and the potential therapeutic effects of aerosolizedhyaluronan (HA) on smoke-induced injury. This agent has previously beenshown to significantly reduce smoke-induced airspace enlargement andprevent elastic fiber injury when given concurrently with smoke¹⁵. Thecurrent investigation modifies the original experimental protocol bydelaying therapeutic intervention for 1 month following initiation ofsmoke exposure, thereby providing a more clinically relevant approach toevaluating this form of treatment. The ability of HA to limit airspaceenlargement and prevent elastic fiber injury, despite pre-existingsmoke-induced lung injury, would support clinical testing of this agentin patients who already have significant evidence of COPD.

Methods

Experimental Plan

Eight-week-old female DBA/2J mice (The Jackson Laboratory, Bar Harbor,Me.) were divided into two treatment groups as follows: Group 1 wastreated with aerosolized HA, beginning 1 month following initiation ofsmoke exposure; Group 2 was treated with aerosolized water, beginning 1month following initiation of smoke exposure. Groups 1 and 2 wereexposed to smoke for 3 h per day, 5 days/week, for a period of 10months. Group 1 was treated with a 0.1% solution of HA in water for 1 hprior to each smoking session. Group 2 received aerosolized water for asimilar interval. At various intervals following initial smoke exposure,animals were euthanized to determine (1) DID content in bronchoalveolarlavage fluid (BALF) and whole lungs at 2, 4, 6, 8, and 10 months; (2)lung histopathology at 3, 6, and 10 months; and (3) airspace enlargementas measured by the mean linear intercept (MLI) at 3 months.

Exposure to Cigarette Smoke

Following administration of either aerosolized HA or water, thenebulizer was disconnected and the smoking machine (Model TE-10, TeagueEnterprises, Davis, Calif.) was attached to the exposure chamber. Bothtreatment groups were exposed to cigarette smoke for a period of 3h/day, 5 days/week. The smoking machine simultaneously burned twofiltered research-grade cigarettes (type 2R4F, University of Kentucky).Each cigarette was puffed once per minute for 2 s at a flow rate of 1.05LPM, yielding 35 cc of smoke. This cycle was repeated nine times beforeejecting the cigarette and loading a new one. Proper flow rate wasmaintained by a vacuum pump that established negative pressure at theexhaust port.

Exposure to HA Aerosol

Beginning 1 month following initial smoke exposure, Group 1 wasadministered a 0.1% solution of low-molecular-weight (150 kDa)streptococcal HA in water (Bayer, Shawnee, Kans.), using a Misty-Oxnebulizer (Vital Signs, Totowa, N.J.). Group 2 received aerosolizedwater alone. The nebulizer was connected to a heavy-duty air compressorthat delivered a constant pressure of 30 psi. The aerosol entered theexposure chamber through an inflow port attached to the roof and wasdrawn through the chamber by negative pressure created by a vacuum pumpconnected to an exhaust port on the side wall. The chamber was largeenough (28×19×15 in.) to permit the mice to remain in their cages whileinhaling the aerosol, thereby minimizing direct handling of the animals.

Light Microscopic Studies

At 3, 6, and 10 months following initiation of smoke exposure, mice wereasphyxiated with CO₂ and their lungs were inflated in situ with 10%neutral-buffered formalin at a constant pressure of 20 cm H₂O. After 2h, the chest contents were removed and fixed for several days informalin. The extrapulmonary structures were then dissected off and thelung tissues were randomly cut and entirely submitted for histologicalprocessing. Slide sections stained with hematoxylin and eosin wereexamined with the light microscope to determine histological changes andto quantify airspace diameter by the mean linear intercept method¹⁶.Additional sections were treated with the Verhoeff-Van Gieson stain toidentify elastic fibers.

Measurement of DID

The levels of the elastin-specific crosslinking amino acids, DID, weremeasured in both BALF and whole-lung tissues. Animals were asphyxiatedwith CO₂ and their lungs were lavaged three times with 1-ml aliquots ofHanks' solution. Both cell-free lavage fluids and homogenized lungtissues were then hydrolyzed in 6 N HCl at 110° C. for 24 h, and thehydrolysates were filtered and evaporated to remove acid. DID werequantified by high-performance liquid chromatography and electrosprayionization mass spectrometry according to previously publishedprocedures¹¹.

Data Analysis

All data were expressed as mean±standard error of the mean (SEM). Thetwo-sample t-test was used to determine statistically significantdifferences between treatment groups.

Results

BALF DID

As shown in FIG. 15, the amount of BALF DID in both the HA/Smoke(Group 1) and Smoke-Only (Group 2) groups dropped precipitously after 4months of smoke exposure. In the HA/Smoke group, there was a decreasefrom 0.8 ng/ml to less than 1 pg/ml during this interval. Whiledifferences in BALF DID between the groups were not statisticallysignificant at 2 and 4 months, subsequent measurements showedsignificantly lower levels of these amino acids in the HA-treatedanimals. Levels of DID were undetectable (<1 pg/ml) in 8-week-oldanimals that were not exposed to either HA or smoke.

Lung DID

The amount of DID in the lungs was also measured at bimonthly intervals,beginning 2 months after the smoking regimen began. Both the HA/Smokeand Smoke-Only groups showed an increase in DID during the first 2months, followed by a decline over the next 4 months and a secondincrease between 6 and 10 months (FIG. 16).

Differences between the groups were not statistically significant overthe entire course of the study.

Lung Histopathology

Exposure to tobacco smoke for 3 months resulted in significant airspaceenlargement in animals receiving smoke alone, whereas only minimalalveolar changes were seen in those treated with HA (FIG. 17). The meanlinear intercept (MLI) of animals treated with HA was 42.6 μm comparedto 58.4 μm for those receiving smoke alone (p<0.01; FIG. 18). There wasno significant difference between the MLI of the HA-treated group andthat of 8-week-old control animals from our previous study (40.5±0.6lm), which were not exposed to either smoke or HA¹⁵. However, animalsreceiving only smoke had a significantly higher MLI than this controlgroup (p<0.01). Similar measurements were not performed at later timeintervals because there was little progression of airspace enlargementin either group, which is consistent with previous morphologicalfindings¹⁵.

Both groups showed inflammation of the larger airways at 3 months,including prominent papillary hyperplasia of bronchial epithelium, andaccumulation of elastic fibers within the connective tissue stalksassociated with the papillary epithelium (FIG. 19). The airwayinflammation persisted at later time intervals (6 and 10 months), butthere was no evidence of alveolitis or interstitial fibrosis in eithergroup, despite the increase in total lung DID between these two timepoints. While several studies have shown that low-molecular-weight HAmay enhance the expression of a variety of cytokines^(17,18), weobserved no evidence of an inflammatory response in the HA-treatedanimals beyond that induced by smoke exposure.

Discussion

The concept of using nebulized HA to prevent elastic fiber injury isbased on a series of experiments designed to determine the potentialrole of agents other than elastases in the pathogenesis of pulmonaryemphysema. Previous studies from this laboratory indicated thatintratracheal instillation of a nonelastolytic enzyme, hyaluronidase,induced pulmonary airspace enlargement in hamsters when administered inconjunction with 60% oxygen¹⁹. Damage to elastic fibers occurred onlywhen both agents were given concomitantly, suggesting the possibilitythat hyaluronidase may facilitate the breakdown of these fibers byincreasing their susceptibility to injury by other injurious agents suchas elastases or oxidants. This concept was supported by subsequent workdemonstrating that pretreatment of the lung with hyaluronidase enhancesairspace enlargement induced by intratracheal administration ofelastase^(20,21).

Studies were then undertaken to examine the effect of HA itself on thismodel of emphysema. Animals were exposed to an aerosol composed of 0.1%HA in water for 50 min prior to intratracheal instillation of elastase.Compared to controls treated with aerosolized water and elastase, thosethat received HA had significantly less airspace enlargement^(22,23).

Although the precise mechanism by which HA prevents lung injury is notyet well understood, our laboratory has shown that HA does not directlyinhibit elastases but instead appears to bind to elastic fibers andprevent elastases from attacking them^(20,22,24). Studies usingaerosolized fluorescein-labeled HA demonstrated preferential adherenceof the polysaccharide to lung elastic fibers^(22,24). This finding wascomplemented by additional experiments in which the binding of HA toelastic fibers in vitro prevented elastolysis by several different typesof elastase, including human metalloproteinase 12, an enzyme that may beresponsible for emphysematous changes associated with cigarettesmoking²².

Interactions between HA and elastic fibers may involve formation ofelectrostatic or hydrogen bonds. The binding sites may not be situatedon the elastin protein itself but may instead be located in thesurrounding matrix composed of microfibrils or other glycoproteins.Alternatively, the exogenously administered HA could combine with nativeHA in close proximity to elastic fibers by a process ofself-aggregation^(26,27). The resulting molecular complexes of HA mayprovide a protective barrier against both free elastases and the cellsthat secrete them¹³.

In contrast to earlier studies in which HA was administeredconcomitantly with cigarette smoke, the current investigation allowedelastic fiber breakdown to proceed unimpeded for the first month, thusproviding a more clinically relevant test of the therapeutic potentialof HA. While concurrent administration of aerosolized HA significantlyreduced BALF DID levels within 3 months of smoke exposure¹⁵, the sameeffect was not seen until 6 months in the present study. Nevertheless,the delay in administering HA did not affect its ability to preventemphysematous changes in the lung.

In the current study, the lack of airspace enlargement in the HA-treatedgroup, despite significant elastic fiber breakdown, may possibly beexplained by the fact that airway injury is an early feature of thismodel of smoke-induced lung injury. Although the precise contribution ofairway inflammation to BALF DID levels remains uncertain, it may bespeculated that the high levels of BALF DID at 2 and 4 months followinginitiation of smoke exposure are a consequence of elastin turnover inthe walls of the larger airways rather than the distal lung.

With regard to total-lung DID, there were no significant differencesbetween the two groups at any time point, suggesting that this parameteris not a sensitive measure of elastic fiber degradation but ratherreflects the balance between elastic fiber injury and repair. Rapidresynthesis of these fibers could mask any differences with regard totheir rate of breakdown.

The development of airway inflammation within the first 2 months ofsmoke exposure may explain why HA was initially ineffective in reducingBALF DID levels. The release of enzymes and oxidants by inflammatorycells may cause the exogenous HA to undergo breakdown, thereby impairingits ability to form larger, protective complexes in proximity to airwayelastic fibers. While such a process remains hypothetical, thislaboratory has previously shown that the same preparation of aerosolizedHA used in the current study is effective in preventing acute lunginjury only when given prior to intratracheal instillation ofendotoxin²⁷.

Whether a similar pattern of elastic fiber breakdown and proliferationoccurs in human lungs in response to smoking remains unclear. However,there is some experimental evidence which suggests that both forms ofinjury have certain features in common. In one study, DID levels inplasma and urine were significantly elevated in COPD patients, with andwithout emphysema, indicating that elastic fiber injury occurs in bothairways and lung parenchyma¹². Other investigators have also reported areduction in urinary desmosine levels as the disease progresses,although their findings were attributed to a loss of lung elastic fibermass rather than a specific decrease in the rate of elastin breakdown²⁸.

The leveling off of airspace enlargement in smoke-exposed mice afterseveral months is consistent with an adaptive response to chronicinjury. A number of studies suggest that enhanced synthesis ofendogenous antioxidants may limit the damaging effects of tobacco smokeand other oxidants²⁹⁻³¹. Furthermore, changes in the interstitialextracellular matrix resulting from continued injury and repair coulddecrease the likelihood of alveolar wall rupture due to elastaseactivity or mechanical stress. Regarding this possibility, an increasein lung collagen content has been reported after prolonged exposure tocigarette smoke, suggesting a transition from a degradative to aproliferative process, similar to that observed in the current study³².

Notwithstanding these limitations, experimental models of smoke-inducedlung injury provide a means of evaluating the usefulness of potentialtherapeutic agents. In the current study, the ability of HA to mitigateboth airspace enlargement and elastic fiber injury, despite a 1-monthdelay in treatment, provides added support for testing this agent inpatients with pre-existing COPD. The gradual progression of this diseasesuggests that even a small decrease in the rate of elastic fiber injurycould have a significant impact on the decline of lung function.

Example 5—Quantitation of Desmosine and Isodesmosine in Urine, Plasma,and Sputum by Tandem Mass Spectrometric Analysis

In this application we describe a practical, and a reliable LC/MSMSanalysis that can measure DES and IDS in all body fluids includingurine, plasma, sputum, and lavages and serve as a standardized method.The analysis utilizes commercially available acetylated pyridinoline asthe internal standard to optimize reproducibility and accuracy.

Materials and Methods

Chemicals

Desmosine (DES) and Isodesmosine (IDS) standard (mixed 50% DES and 50%IDS) were purchased from Elastin Products Company (Owensville, Mich.).Acetylated pyridinoline was obtained from Quidel (San Diego, Calif.).CF1 cellulose powders were purchased from Whatman (Clifton, N.J.), andall other reagents were from Sigma (St. Louis, Mo.).

Sample Collection and Human Subjects

Urine (24 hour samples), plasma, and sputum samples were collected aspreviously described^(11,54) from volunteers with informed consent atthe James P. Mara Center for Lung Disease, St. Luke/Roosevelt HospitalCenter, New York. COPD was diagnosed in study patients according to theGlobal initiative for Chronic Obstructive Lung Disease grades 1 to 4⁶⁰.Patients gave informed consent for the study. Control subjects wereselected by clinical history free of any specific known disease orsignificant symptoms, and none have ever smoked. The study was approvedby the Institutional Review Board.

Creatinine and Protein Measurement

Urine creatinine was measured by the commercially available 555Acreatinine kit (Sigma-Aldrich). Total protein in plasma and sputumsamples was measured by the commercially available microprotein assaykit (Sigma-Aldrich), which is based on protein-dye (Coomassie blue)binding.

Acid Hydrolysis

Samples of urine (0.1 ml), plasma or sputum (0.5 ml each) were placed ina glass vial with equal volumes of conc. HCl (37%). Air in the vial wasdisplaced with nitrogen, and was heated at 110° C. for 24 hrs. Thehydrolyzed sample was filtered and evaporated to dryness. For the free(unconjugated) forms of DES and IDS analysis, 0.2 ml of urine wasanalyzed directly without the HCl hydrolysis.

Cellulose (CF1) Cartridge Extraction

The acid hydrolyzed samples (after drying under vacuum or nitrogenstream to remove residual acid) or unhydrolyzed urine sample (for freeDES and IDS analysis) were treated with 1 ng (for urine samples) or 0.5ng (for plasma and sputum samples) of acetylated pyridinoline as theinternal standard.

The mixture was dissolved in 2 ml of n-butanol/acetic acid/water(4:1:1), and applied onto a 3 ml cellulose cartridge, which was preparedby introduction of 3 ml of 5% CF1 cellulose powder slurry inn-butanol/acetic acid/water (4:1:1). The cellulose powder slurry must bea well dispersed slurry by stirring for 24 hrs. The cartridge was washed3 times with 3 ml of n-butanol/acetic acid/water (4:1:1), and thesamples retained in the cartridge were eluted with 3 ml of water, driedand dissolved in 200 μl (for urine sample) or 100 μl (for plasma andurine samples) of LC mobile phase and analyzed by LC/MSMS.

LC/MSMS Analysis

A TSQ Discovery electrospray tandem mass spectrometer (Thermo FisherScientific) was used for LC/MSMS analysis.

HPLC conditions used were a 2 mm×150 mm dC18 (3 μm) column (Waters,Mass.) and the mobile phase A (7 mM HFBA/5 mM NH₄Ac in water) and B (7mM HFBA/5 mM NH₄Ac in 80% acetonitrile) were programmed linearly from100% A to 82% A in 12 mins.

Quantitation was performed by selected reaction monitoring (SRM) of thetransitions of both DES and IDS (m/z 526 to m/z 481+m/z 397) and theinternal standard (m/z 471 to m/z 128), with collision energy set at 33V for both transition, collision gas pressure was 1.5 mTorr, tube lensat 132 V, with sheath gas pressure set at 45 and auxiliary gas pressureat 6 units and ion spray voltage at 3.8 kV. The scan time set at 1.00msec and both quadrupoles (Q1 and Q3) were at 0.7 Da FWHM.

Statistical Analysis

A t-test adjusted for unequal variance was used to test the nullhypothesis. The level of significance was 0.05. The p-values werecalculated based on the summed values of DES and IDS using the unpairedt-test (The used software is “GraphPad Prism 4 (2)”).

Results

DES and IDS are Stable Toward HCl Hydrolysis

The assay of total DES and IDS in biological fluids requires HClhydrolysis at 110° C. to release DES and IDS from their crosslinked orpeptide conjugates. We examined the stability of DES and IDS in threedifferent concentrations (10, 5, and 1 ng/ml) during HCl hydrolysis at110° C. for 24 hrs. The DES/IDS solutions resulting from the HCltreatment were subjected to LC/MSMS measurements of DES and IDS, whichwere compared with the measurements of the same concentrations ofuntreated DES and IDS to calculate the recovery by the acid treatment.The results show that DES and IDS are stable through acid hydrolysiswith virtually complete recoveries at all three concentrations (Table3).

Recovery of DES and IDS in Human Body Fluids

Three known amounts of DES/IDS were spiked into the control urine,plasma, and sputum samples at concentration ranges expected to beencountered in biological samples (10, 20 and 40 ng/ml in urine samples;0.2, 0.4 and 0.8 ng/ml in plasma and sputum samples). The mixtures weresubject to HCl hydrolysis at 110° C. for 24 hrs, addition of IS, CF1cartridge chromatography, and LC/MSMS analysis under the establishedprocedure to measure DES/IDS levels. The recoveries of DES/IDS are above99% in urine, 94% in plasma, and 87% in sputum samples; with theimprecision 8%, 9%, and 10%, respectively (Table 2). Limit ofquantitation (LOQ) in all relevant body fluids are determined as 0.1ng/ml of samples based on reproducibility and recovery study (Table 4).

Acetylated Pyridinoline as Internal Standard

Acetylated pyridinoline is an acetylated derivative of 3-hydroxypyridinoline which serves as a trifunctional crosslink in collagen. Thecompound has been used as an internal standard (IS) in HPLC analysis ofpyridinium crosslinks of collagen in urine or tissue⁶¹⁻⁶³.

Since acetylated pyridinoline has a similar molecular structure andpolarity to that of DES and IDS (FIG. 20A), the use of the pyridinolineas an IS for the LC/MSMS analysis of DES and IDS was explored. Theelution characteristics of acetyl pyridinoline in both a CF1 cartridgeand HPLC column are closely similar to DES and IDS (FIGS. 11 and 20B).It also exhibits a similar linear response in mass spectrometricanalysis. Since DES and IDS have been found stable without lossesthrough HCl hydrolysis, we introduce acetylated pyridinoline as IS afterthe HCl hydrolysis for the DES and IDS quantitation to determinerecovery from the subsequent analytical procedure. The linear responsesof DES and IDS using acetylated pyridinoline as the IS for theanalytical procedure are shown in FIGS. 21A-B. As also shown in FIG. 12and Example 3, our HPLC chromatography can effectively separate the twoDES and IDS isomers; thus, the isomers can be conveniently quantifiedseparately using the calibration curve of FIG. 21A or as DES+IDS usingthe calibration curve of FIG. 21B.

Standardized LC/MSMS Analysis of DES and IDS in all Body Fluids

We have developed the following conventional three-step analyticalprocedure which can be used to measure DES and IDS in all relevant bodyfluids (urine, plasma, and sputum) or lavage fluids:

1. Sample hydrolysis: To body fluid samples (0.1 ml urine, 0.5 ml plasmaor sputum) are added with equal volumes of concentrated HCl (e.g., about6-12N) and heated in nitrogen at 110° C. for 24 hours. (For free urinaryDES and IDS analysis, this hydrolysis step is eliminated)

2. CF1 cartridge extraction: Add 1 ng (for urine) or 0.5 ng (for plasma,sputum) of internal standard (acetylated pyridinoline) and apply to a 3ml cellulose cartridge prepared by introduction of 5% CF1 cellulosepowder slurry in n-butanol/acetic acid/water (4:1:1), wash the cartridgewith n-butanol/acetic acid/water (4:1:1), and elute the analytes outwith 3 ml of water.

3. LC/MSMS analysis: HPLC separation by a 2 mm×150 mm dC18 (3 μm) columnand measure DES and IDS by SRM monitoring of transition ions; DES or IDS(m/z 526 to m/z 481+397) and IS (m/z 471 to m/z 128).

Measurement of DES and IDS in Urine, Plasma, and Sputum of COPD Patientsand Healthy Subjects

The analytical procedure we have developed was used to compare thelevels of DES and IDS in urine, plasma, and sputum obtained from COPDpatients and that of healthy normals. The results are shown with urinesamples in FIG. 22A, plasma samples in FIG. 22B, and sputum samples inFIG. 22C. The urinary DES/IDS levels after HCl hydrolysis (totalDES/IDS) in COPD patients are slightly higher than that of healthycontrols, but the difference is not statistically significant(p=0.1942). On the other hand, the urinary DES/IDS levels without HClhydrolysis (free DES/IDS) in COPD patients are statisticallysignificantly higher than that of healthy controls (p=0.0094). Theratios of free DES/IDS to total DES/IDS are significantly higher in COPDpatients than that of controls (p<0.0001) (FIG. 22A). This result is inagreement to our previous data (23,24). Also the data indicates that theratio of total DES/IDS to free DES/IDS levels in urine samples are goodbiomarkers for showing increased elastase activity in COPD patients. Inplasma, DES/IDS levels in COPD patients are statistically higher thanthat of the healthy controls (p<0.0001) (FIG. 22B). The DES/IDS insputum of COPD patients shows the median of 0.24 ng/ml, while the levelsin induced sputum from healthy subjects are below detection limit (0.04ng/ml) (FIG. 22C).

Discussion

A strength of DES/IDS as biomarkers in COPD is the recognition thatmatrix elastin is a structural target of the disease. Among the manytechniques developed to measure DES/IDS, the LC/MSMS technique whichprovides higher sensitivity and specificity appears to be an improvedchoice for biomarker analysis⁵⁵. Several modifications of LC/MSMSmethods for DES/IDS analysis have been reported recently, but they areall developed for the measurements of DES/IDS in urine⁵⁶⁻⁵⁸. Ourprevious studies on the measurement of DES/IDS in COPD patients^(11,54)and DES/IDS levels in response to Tiotropium treatment of COPDpatients¹⁰ demonstrate that DES/IDS levels in sputum and plasma areeffective indicators of elastin degradation in patients in the body as awhole and the lung per se. Development of a sensitive, accurate, andreproducible method which can measure DES/IDS levels in all body fluidscan be clinically meaningful especially related to parameters such aslung structure analyzed by computed tomography quantitative, lungfunction or genomic analysis.

Previously published LC/MS or LC/MSMS methods^(11,54-58) all havedisadvantages of either lack of a reliable internal standard or themethod not standardized for the analysis of all relevant body fluids(i.e., urine, plasma, and sputum), which are important for assessingclinical meaning of elastin degradation. In this application we havedeveloped a practical and simplified LC/MSMS analytical procedure thatcan be universally utilized for the analysis of DES and IDS in allrelevant body fluids; including urine, plasma, and sputum.

We confirm that DES/IDS are stable under the conditions of hydrolysis in6N HCl at 110° C. for 24 hours, the acid hydrolysis generally used torelease DES/IDS from their peptide conjugate (Table 3). We introduceacetylated pyridinoline as the internal standard after the HClhydrolysis step to correct for losses occurring from the subsequentsteps. Acetylated pyridinoline has a closely similar molecular structureand chromatographic mobility to that of DES and IDS molecules, whichenables the development of a reproducible and accurate LC/MSMSmeasurement of DES/IDS in urine, plasma, and sputum. The LC/MSMSanalysis can also effectively separate the two DES/IDS isomers (FIG.20B), thus two DES/IDS isomers can be conveniently quantified eitherseparately (FIG. 21A) or as combined DES+IDS (FIG. 21B) in all bodyfluids.

TABLE 3 Stability of Desmosine (DES) and Isodesmosine (IDS) on acidhydrolysis* 10 ng/ml DES/IDS 5 ng/ml DES/IDS 1 ng/ml DES/IDS Recovered(ng/ml) DES IDS DES/IDS DES IDS DES/IDS DES IDS DES/IDS Means 5.04 5.3710.41 2.42 2.55 4.98 0.50 0.48 0.98 SD 0.32 0.30 0.61 0.20 0.20 0.240.05 0.05 0.10 % 6 5 6 8 8 5 10 11 10 % Recovery 101 107 104 97 102 100100 96 98 *DES/IDS of three concentrations were hydrolyzed in 6N HCl at110° C. for 24 hour, after addition of internal standard DES/IDS werere-isolated by SPE (CF1 column) chromatography, and quantified byLC/MSMS analysis

Two previous reports have introduced deuterated compounds as internalstandards to improve LC/MSMS analysis of DES/IDS^(57,58). However theorigin of the deuterium compounds are not stated, and appeared to beobtained through catalytic proton exchange reactions with DES/IDS. Thestructures and the stability of the introduced standard were notdemonstrated. In this regard, acetylated pyridinoline is a commerciallyavailable compound with defined structure, which can be readily added asthe internal standard. The accuracy and reproducibility of the developedLC/MSMS analysis was further tested by recovery studies of DES/IDS in aseries of known contents of DES/IDS in urine, plasma, and sputum samplesas shown in Table 4. The recoveries from urine, plasma, and sputumsamples are above 99%, 94%, and 87%, respectively, with goodreproducibility.

TABLE 4 Recovery of Desmosine (DES) and Isodesmosine (IDS) from Urine,Plasma, and Sputum* Recovery from Urine Control Urine Add 40 ng/ml urineAdd 20 ng/ml Urine Add 10 ng/ml Urine Recovered (ng/ml) DES IDS DES/IDSDES IDS DES/IDS DES IDS DES/IDS DES IDS DES/IDS Mean(n = 3) 3.82 3.247.06 23.3 23.33 46.65 14.14 14.36 28.5 8.81 8.2 17.01 ±SD 0.29 0.66 0.941.87 2.01 3.86 0.41 2.06 2.44 0.87 0.75 1.61 % CV 8 20 13 8 9 8 3 14 910 9 9 % Recovery*2 98 100 99 102 109 105 100 100 100 Recovery fromPlasma Control Plasma Add 0.8 ng/ml plasma Add 0.4 ng/ml plasma Add 0.2ng/ml plasma Recovered (ng/ml) DES IDS DES/IDS DES IDS DES/IDS DES IDSDES/IDS DES IDS DES/IDS Mean(n = 3) 0.23 0.13 0.35 0.57 0.57 1.15 0.40.33 0.72 0.31 0.22 0.52 ±SD 0.02 0.03 0.04 0.02 0.03 0.05 0.02 0.040.03 0.05 0.03 0.05 % CV 7 22 12 4 6 4 4 12 4 15 12 10 % Recovery*2 91109 99 93 100 96 94 95 94 Recovery from Sputum Control Sputum Add 0.8ng/ml Sputum Add 0.4 ng/ml Sputum Add 0.2 ng/ml Sputum Recovered (ng/ml)DES IDS DES/IDS DES IDS DES/IDS DES IDS DES/IDS DES IDS DES/IDS Mean(n =3) 0.09 0.03 0.12 0.4 0.4 0.8 0.24 0.24 0.48 0.19 0.12 0.32 ±SD 0.010.01 0.01 0.06 0.05 0.09 0 0.04 0.04 0.04 0.02 0.01 % CV 13 42 7 15 1111 0 18 9 18 18 4 % Recovery*2 81 94 87 84 104 93 101 95 99 *1) DES/IDSwere spiked into control body fluids at three concentrations (expectedranges of detection). The samples were acid hydrolyzed (6N HCl at 110°C. for 24 hrs), chromatographed by SPE (CF1 column). After addition ofthe internal standard DES/IDS were measured by LC/MSMS and calculatedfor recovery. *2) More accurate % Recovery was obtained by integrationof combined areas of DES/IDS. % Recovery of individual DES and IDS mayvaried by chromatographic separation.

This proposed method was used to measure DES and IDS in urine, plasma,and sputum of a cohort of COPD patients as compared to their healthycontrols (FIG. 22A-C.) The results confirm our previous reports⁵⁴ thatthe DES/IDS levels are useful biomarkers to characterize elastindegradation in COPD.

The degradation of elastin-containing tissues also occur inaorta^(44,64), skin^(43,65,66), and liver⁶⁷, etc. The developed LC/MSMSanalysis of DES/IDS can have wide application for investigating diseaseswhich involve in those elastic tissues.

In sum, we have developed a sensitive, reproducible, and practicalmethod using tandem mass spectrometric LC/MSMS analysis to measure DESand IDS using acetylated pyridinoline as the internal standard. Thisprocedure can serve as a standardized LC/MSMS method to measure DES andIDS in all relevant body fluids, which are important for the clinicalassessment of elastin degradation in diseases. The developed methoddemonstrated increased DES/IDS levels in urine, plasma, and sputumsamples of patients with COPD over healthy controls. This analyticalmethod can be applied to investigate diseases which induce elastictissue degradation in vivo.

Example 6—The Effect of Second Hand Smoke Exposure on Markers of ElastinDegradation

As set forth above, desmosine and isodesmosine (D/I) are two crosslinkedpyridinoline amino acids specific to peptides produced from elastindegradation⁵⁵. D/I have been measured by liquid chromatography tandemmass spectrometry (LC/MS/MS) in patients with COPD and found to beelevated as compared to normal controls. For the first time thesepeptides have been measured in subjects exposed to second hand smoke.Desmosine and isodesmosine were found to be statistically significantlyelevated in patients exposed to second hand smoke as compared to normalcontrols.

Materials/Methods

Patients

Two cohorts of subjects, Cohorts I and II, were studied. Cohort I hadthree sub-groups of subjects which were studied for the effect of SHSexposure on D/I levels. These subjects were part of an ongoing study todetermine the effect of SHS exposure on hormonal constituents infemales. All subjects completed a lifestyle and nutritionalquestionnaire that included a description of exposure to cigarettetobacco smoke. Subjects were divided into three groups, active smokers,passive smokers and non-exposed. Passive smokers were defined as anyonewho has lived with or has been exposed to cigarette smoke on a dailybasis, but were not smokers. Most subjects were exposed within the home.

Active smokers were persons who were smoking daily. Exclusion criteriaincluded several medical conditions including: congestive heart failure,myocardial infarction, cerebral vascular accident, asthma, bronchitis,emphysema, any malignancy; also subjects exposed to dyes (textiles, artsand crafts) on a regular basis. Subjects were initially screened aseligible by telephone and then came for a study visit. All patients werefemale between the ages of 18-50 yrs. with the majority of them in thelate twenties and thirties. They were not taking hormonal contraceptivesand were not pregnant at the time of the study.

Cohort II was also subdivided into 3 groups of active smokers, passivesmokers and non exposed in smaller numbers. Subjects were males andfemales between the ages of 22 and 69 years. They were recruited from amedical clinic in the Veteran's Administration Hospital where, afterclinical evaluation, they were considered to be in normal health andwere, or were not, exposed to cigarette smoke. Some subjects respondedto an advertisement requesting participation in this study. Mostsubjects were exposed to second hand smoke in the occupational setting;i.e. bartenders, construction workers, office workers. All passive smokeexposures in Cohorts I and II were current and not past.

The degree of second hand smoke exposure in Cohort I was determined fromthe volunteered histories given on a questionnaire. Subjects reportedthe number of individuals that smoked in the household, an estimation ofthe number of cigarettes each individual smoked per day and the periodtime the subject lived in the household. A number was calculatedreflecting these variables. A score less than 2,000 was considered mildexposure; 2,000-10,000 moderate exposure and over 10,000 severeexposure. Mild, moderate and severe exposures were then used in theanalysis.

Such detailed information on the occupation and household environmentalexposure was not available in Cohort II. The subjects of Cohort IIindicated their occupation and that they were exposed to second handsmoke and were not actively smoking themselves.

Chemicals

Desmosine (D) and Isodesmosine (I) standard (mixed 50% D and 50% I) werepurchased from Elastin Products Company (Owensville, Mich.), CF1cellulose powders were from Whatman (Clifton, N.J.), and all otherreagents were from Sigma (St. Louis, Mo.).

Preparation of Blood Samples

Plasma samples were obtained after centrifuging venous blood specimenswith EDTA for 2,500 revolutions per minute for 15 min. Plasma sampleswere stored at −80° C. until used. 0.25 ml of serum and 0.25 mL ofconcentrated HCl (37%) were placed in a glass vial. After air in thesample was displaced with a stream of nitrogen, the sample was acidhydrolyzed at 110° C. for 24 hr. The hydrolysates were filtered anddried, the residue was dissolved in 2 mL of the mixed solution(n-butanol/acetic acid/water, 4:1:1 by volume). The sample solution wasloaded onto a 3-mL CF1 cartridge. The CF1 cartridge was prepared byintroducing 3.5 mL of the slurry of 5% CF1 cellulose powder in the mixedsolution between two polypropylene frits. The cartridge was washed threetimes with 3 mL of the mixed solution, and the D and I adsorbed in theCF1 cartridge were eluted with 3 mL of water. The eluate was evaporatedto dryness under vacuum at 45° C., and the residue was dissolved in 0.1mL of HPLC mobile phase for LC/MS/MS analysis.

Samples were processed and measured in duplicate, and the results wereaveraged. The mean recoveries of D/I and from 0.20 ng/ml plasma were67±1 and 72±4%, respectively. Values in plasma were corrected forrecovery losses.

Measurement of Desmosine/Isodesmosine by LC/MS/MS Analysis

The Thermoscientific TSQ Quantum Discovery tandem LC/MS/MS system wasused for the analysis. HPLC column was a 150×2 mm (3 μm) Atlantis dc18(Waters, Miss.). The mobile phase A is a solution containing 5 mMammonium acetate and 7 mM heptafluorobutyric acid in water and themobile phase B is a solution containing 5 mM ammonium acetate and 7 mMheptafluorobutyric acid in a acetonitrile/water (8:2 ratio). The flowrate of mobile phase is 0.2 ml/min and is programmed from mobile phase A100% to 88% in 12 mins.

Reaction ion monitoring (RIM) of the transition ions, m/z 526 to m/z481+m/z 397, was used for the quantitative measurements of D and I.Between-run imprecision (% CV) at D/I levels of 0.10, and 0.20 ng/mlwere 4.0% and 3.9% respectively.

IRB approval has been obtained from IRB Georgetown University MedicalCenter (2006-132) and from Carl T. Hayden, VAMC (Robbins 003).

Measurement of Cotinine by LC/MS/MS Analysis

To indicate exposure to cigarette tobacco smoke, plasma cotinine, ametabolite of nicotine, was quantitated using LC/MS/MS (AppliedBiosystem API-4000 or Thermoscientific TSQ Quantum Discovery) followingextraction by a solvent (ethyl acetate) extraction. Internal standard(D₃-cotinine) solution was added to subject samples and samples werevortexed and centrifuged. The supernatant was injected into the LC/MS/MSsystem. Cotinine was measured by monitoring the Q1/Q3 transition ions ofcotinine at m/z 177 to 80 and D₃-cotinine at m/z 180 to 80. Between-runimprecision (% CV) at cotinine levels of 33, 124, and 248 ng/ml were7.2, 5.6, 3.6% respectively.

Statistical Analysis

A t-test adjusted for unequal variance was used to test the nullhypothesis. The level of significance was 0.05. The p-values werecalculated based on the summed values of D and I using the unpairedt-test (The used software is “GraphPad Prism 4 (2)”).

Results

Results for all three groups of non-exposed, exposed, and smokers in 98subjects (Cohort I) are shown in FIG. 23A-B. The mean D and I level innon-exposed is 0.22±0.07 ng/ml (n=30), while the level in exposed is0.29±0.10 ng/ml (n=34) and smokers is 0.34±0.12 ng/ml (n=34). The pvalue when comparing non-exposed D and I levels to exposed wasstatistically significant, P=0.005. The p values of non-exposed tosmokers was <0.0001, and the value of exposed vs. smokers is nearstatistical significance, p=0.0533 (FIG. 23A).

Cohort II compared D/I plasma levels and levels of cotinine also in 22subjects of non-exposed, exposed, and smokers. The study includes malesas well as females and shows the D and I levels and cotinine levels inthose subjects (FIGS. 24A-24B). The average D/I (ng/ml plasma) was0.21±0.03 in the non exposed group and 0.40±0.13 in the second handsmoke exposed group. The smoking group had a measurement of 0.55±0.28.The p values were 0.0233 for the non-exposed vs. the second hand smokeexposed group and 0.0075 for the non-exposed vs. smoking group. Theresulting p value when second hand exposure was compared to activesmoking was not significant 0.1782 (FIG. 24A).

Cotinine values for each of the cohorts are shown in FIGS. 23B and 24B.Cotinine values showed a large variation in each cohort. In Cohort Ithere was a significant correlation between the plasma level of D/I andthe log value of plasma cotinine as shown in FIG. 25. In Cohort II, withfewer subjects overall and fewer in each category of exposure, suchcorrelations were not statistically significant. Cotinine levels inCohort I in second hand tobacco smoke exposure exceeded those in cohortII while D/I levels were higher in Cohort II second hand smokers than inCohort I.

Comparison of the mean levels of plasma D/I and cotinine in Cohorts Iand II showed no statistically significant difference in D/I in thenon-exposed group but statistically significant differences in theexposed and actively smoking groups (p=0.023 and p=0.004), respectively,for D/I but not statistically significant differences for cotinine forthose groups respectively (p=0.350 and p=0.560).

Among active smokers the levels of D/I were higher in Cohort IIconsistent with their higher pack/year histories.

Discussion

The United States EPA estimates that passive smoking accounts forroughly 3000 deaths in the United States secondary to lung cancerannually⁶⁸. The impact of passive smoking on overall health is clearlyrecognized as an independent risk factor for diseases including thelung⁷¹.

The levels of D and I in urine and plasma in COPD have been found to beconsistently elevated in many studies by various analyticalmethods^(54,55). This raises the prospect that elevated levels of D/Iinduced by second hand smoke exposure become an indicator of risk todevelop COPD in later life.

As shown above, both cohorts of subjects demonstrate a statisticallysignificant increase in plasma levels of D/I in subjects exposed to SHSnone of whom had clinical symptoms of respiratory disease.

Disclosed herein are the first reported measurements of the effect ofsecond hand smoke exposure on elastin degradation in human subjects.This is a demonstration of a tissue matrix effect which may have specialsignificance for lung structure. The further implication of increasedelastin degradation of mature elastin is increased elastase activity intissues and possibly an upgraded inflammatory state. The patientsstudied in all groups were not symptomatic. Neither group had anysignificant medical history or evidence of lung pathology. All hadequivalent and average exercise tolerance. As is the case with theeffects of direct smoke exposure which takes years before evidence ofCOPD is apparent, SHS smoke exposure may produce its effects decadeslater. The results also suggest that even mild passive smoke exposurecould have deleterious effects on lung parenchyma, which if persistentlong term may result in parenchymal inflammation and destructionresulting in COPD.

The clinical significance of the findings in this study are stillunclear. As mentioned earlier, patients with excessive, chronic secondhand smoke exposure do suffer the sequalae of active smokers⁷⁰. Inpatients with COPD, elastin fibers have been shown to be degraded anddisorganized, and the content of lung elastin is low in suchpatients^(45,50). Also once elastin breakdown has occurred, theresulting peptides may promote further parenchymal destruction sincethey may be antigenic and chemotactic. The peptides are chemotactic forneutrophils and macrophages, resulting in a chronic inflammatory statewhich could induce further elastin degradation and possibly clinicalsequelae in the long term⁵¹.

The increase in levels of D/I in plasma of subjects undergoing SHSexposure raises the question of the mechanism for the increase. SinceSHS enters via the respiratory tract the initial effect should be onlung cells and most notably neutrophils and macrophages which undergostimulation with increased synthesis and secretion of elastases. Thisaugmented activity of elastolysis then can effect degradation of elastinin any site in addition to the lung such as blood vessels and skin whichwould elevate plasma concentrations.

D and I as biomarkers for COPD have been studied for some time⁵⁵. Withadvances in technology and preparation of samples, they can be detectedwith greater sensitivity and specificity⁵⁴. This is significant sincethe levels in plasma of patients exposed to second hand smoke may havebeen previously too low to detect. Plasma can be easily obtained frompatients. In the past, 24 hr urine has been studied as a medium for Dand I measurement. Though promising since there is an increase in free Dand I levels in urine in active smokers and COPD patients, it is oftendifficult for the patient and the practitioner to accurately collect 24hr urine⁵⁴. If we are able to accurately and precisely detect increasedlevels in plasma it brings us possibly one step closer to a practicalmarker of elastin degradation and in effect one step closer tounderstanding and quantifying lung degradation.

It would be clinically important to learn what happens to the levels ofD and I over time in these individuals and whether the levels decreaseif the patients are removed from passive smoke exposure, and if they arenot removed if the levels continue to increase in response to increasedelastin degradation. Menzies et al previously illustrated that when theinciting factor was removed; i.e. smoke in bars where they worked, thesubjects rapidly returned to their previous state of health⁷⁴. Suchfindings would suggest that the levels of D and I should decrease whensecond hand smoking is terminated. The clinical implications on lungfunction that this early degradation imposes are unclear. It isnoteworthy that the levels of D and I, in passive smokers, were not ashigh as seen in patients with COPD or alpha-1 antitrypsin deficiency⁵⁴.This finding is not surprising given the burden of disease in patientswith COPD and alpha-1 as compared to our study population.

Certain methodological limitations should be noted in this study. Thedata is reliant on an accurate recognition of second hand smoke exposureby our subjects through self-reporting. This is based on an extensivequestionnaire characterizing their exposure to second hand smoke. Inprevious studies a good correlation between a person's perception ofsecond hand smoke and the lab findings consistent with exposure to SHShas been shown⁷³. The questions are very specific about who smokes inthe home, how much, for how long, and how much time the subject spendswithin this environment. The questionnaire administered to all personsparticipating in this study included the same questions, therefore eachstudy group was exposed to the same level of bias.

Of interest is the measurement of cotinine levels. Previous studies haveestablished a positive correlation between the level of SHS exposure andinflammatory cytokine elevation^(77,82). On average they weresignificantly elevated in subjects that self-reported SHS exposure whencompared to subjects without any exposure. The drawback is that cotinineremains elevated only for short periods of time post exposure andcorrelations with presumed exposure may be weak⁸³. This could explainwhy certain subjects have low cotinine levels.

In the past, multiple studies illustrated that the levels ofinflammatory cytokines, on average, are higher in males thanfemales^(70,72,74). It is postulated that males are more sensitive tothe inflammatory cytokines than females vs. a possibility of greaterexposure in males than in females. It is noteworthy that second handsmokers in Cohort I, which was exclusively a female population ofsubjects, has lower levels of D/I for the cotinine level which washigher than that among passive smokers in Cohort II. This raises thepossibility that females may be less susceptible to elastin degradationby exposure to tobacco smoke, a prospect which should be exploredfurther.

This study illustrates that second hand smoke exposure may be asdangerous and harmful as active smoking on tissue matrix injury andpossibly lung parenchyma. This study demonstrates that D and I can beused to detect early changes of elastin degradation, before clinicallysignificant symptoms occur, and possibly to indicate progression of thedisease. Thus, D and I can serve as biomarkers of exposure. It would besignificant to determine, if increased levels of D and I can becorrelated with computed tomography (CT) findings of significantparenchymal destruction, before clinical symptoms occur. Overall havinga tissue matrix component effected by second hand smoke and detectableby chemical analysis is a useful adjunct to evaluating clinical andphysiological consequences of environmental smoke exposure.

Example 7—Characterization of Peptide Fragments from Lung ElastinDegradation in Chronic Obstructive Pulmonary Disease

As noted above, COPD is characterized by destruction of alveolar walls,obstruction of bronchioles, and trapping of air^(86,87). An earlyinsight into the mechanisms leading to alveolar destruction in patientswith pulmonary emphysema is that lung matrix elastin is a target forprotease degradation by cellular elastases^(80,88,89).

Lung elastin is a highly cross-linked insoluble protein formed bycondensation of lysyl residues in the soluble precursor, tropoelastin(786 amino acids; Table 5), which can be degraded into soluble peptidefragments by the elastolytic enzymes produced by neutrophils andmacrophages. Degradation of lung elastin occurs in COPD as well as insmoking subjects and has been investigated by measurement of 2lysyl-derived cross-linked pyridinium molecules, desmosine andisodesmosine^(11,14,40,54,90), which only exist in elastin and have beenshown to be useful biomarkers for lung elastin degradation in COPDpatients^(54,55).

TABLE 5  Human Tropoelastin Sequence (Swiss-Prot P15502) (SEQ ID NO: 1)MAGLTAAAPR PGVLLLLLSI LHPSRPGGVP GAIPGGVPGG VFYPGAGLGA 50LGGGALGPGG KPLKPVPGGL AGAGLGAGLG AFPAVTFPGA LVPGGVADAA 100AAYKAAKAGA GLGGVPGVGG LGVSAGAVVP QPGAGVKPGK VPGVGLPGVY 150PGGVLPGARF PGVGVLPGVP TGAGVKPKAP GVGGAFAGIP GVGPFGGPQP 200GVPLGYPIKA PKLPGGYGLP YTTGKLPYGY GPGGVAGAAG KAGYPTGTGV 250GPQAAAAAAA KAAAKFGAGA AGVLPGVGGA GVPGVPGAIP GIGGIAGVGT 300PAAAAAAAAA AKAAKYGAAA GLVPGGPGFG PGVVGVPGAG VPGVGVPGA 350IPVVPGAGIP GAAVPGVVSP EAAAKAAAKA AKYGARPGVG VGGIPTYGVG 400AGGFPGFGVG VGGIPGVAGV PSVGGVPGVG GVPGVGISPE AQAAAAAKAA 450KYGAAGAGVL GGLVPGPQAA VPGVPGTGGV PGVGTPAAAA AKAAAKAAQF 500GLVPGVGVAP GVGVAPGVGV APGVGLAPGV GVAPGVGVAP GVGVAPGIGP 550GGVAAAAKSA AKVAAKAQLR AAAGLGAGIP GLGVGVGVPG LGVGAGVPGL 600GVGAGVPGFG AGADEGVRRS LSPELREGDP SSSQHLPSTP SSPRVPGALA 650AAKAAKYGAA VPGVLGGLGA LGGVGIPGGV VGAGPAAAAA AAKAAAKAAQ 700FGLVGAAGLG GLGVGGLGVP GVGGLGGIPP AAAAKAAKYG AAGLGGVLGG 750AGQFPLGGVA ARPGFGLSPI FPGGACLGKA CGRKRK 786

The degradation of cross-linked elastin by elastases results in elastinfragments of varying molecular weight and can be identified aselastin-derived peptides (EDPs). Immunologic detection of EDPs in bodyfluids in COPD patients and smokers has been studied, but provideslittle information with regard to their structuralidentities^(38,92,93). Extensive purifying and sequencing studies ofelastin molecules in the elastic fibers in bovine ligament or aorta havebeen carried using various proteolytic enzymes^(33,94-100). However, thecross-linking domains and structure have not been fully characterizedbecause of the protein's high degree of cross-linking and itsinsolubility. Using liquid chromatography/tandem mass spectrometry(LC/MSMS) analysis, Barroso et al.¹⁰⁰ reported the amino acid sequencesin the peptide fragments produced by different proteolytic enzymes invitro, but did not further study the detection of such peptides in bodyfluids. In the present application we utilize LC/MSMS analysis tocharacterize a full spectrum of EDPs obtained in vitro with 2representative elastases, human neutrophil elastase (HNE) and macrophagemetalloproteinase (MMP12), which have been involved clinically in lungelastin degradation^(7,101). We further demonstrate the detection ofsome of the characterized peptides in the body fluids of patients withCOPD.

The structural characterization of EDPs detected in patients canidentify the enzymatic reactions leading to their formation and theirpossible role in pathogenesis. In addition, EDPs have been shown to playa role in cellular behavior within the extracellular matrix, such aschemotaxis for neutrophils and macrophages or tumor cells. Such factorsmay affect the progression of COPD or pulmonary metastasis once elastindegradation has occurred¹⁰²⁻¹⁰⁵. Antigenic autoimmune effects of EDPs inCOPD patients have also been demonstrated^(106,107).

Materials and Methods

Materials

Purified human lung elastin, bovine neck ligament elastin, human sputumneutrophil elastase (HNE), murine macrophage metalloproteinase (MMP12)were obtained from Elastin Products Company (Owensville, Miss.). Porcinepancreatic elastase (PPE) was purchased from Worthington (Lakewood,N.J.), and synthetic peptide standards were obtained from GenScript(Piscataway, N.J.).

Patients Studied

The 5 patients studied were selected at random from subjects with adiagnosis of COPD (Table 6). The diagnostic criteria conformed to thosestated in the Global Criteria and Guidelines⁶⁰. One of the 5 patientshad α1-antitrypsin deficiency (AATD) of the ZZ phenotype. All had astrong smoking history from 30 to 79 pack-years but had stopped smokingapproximately 3 years prior to this study. Four healthy control subjectswere males, ages 35 to 85, in good health without respiratory symptomsor a history of smoking or exposure to second hand smoke.

TABLE 6 Patients With Diagnosis of COPD Smoking FVC FEV₁ RV/TLC DLCOhistory D/I Patients Age Gender Race % Pred % Pred % Pred % Pred (Pk-Yr)BMI (ng/mL)  1* 42 M C 70 23 34 64 79 30.4 0.52 2 68 F H 67 46 81 98 1823.1 0.51 3 63 M C 74 46 59 59 50 22.1 0.32 4 82 F C 57 41 — — 60 21.00.54 5 88 M C 99 68 65 54 30 23.6 0.66 Note. FVC % Pred = forced vitalcapacity % of predicted; FEV₁% Pred = forced expiratory volume in 1second % of predicted; RV/TLC % Pred = residual volume/total lungcapacity % of predicted; BMI = body mass index; D/I = plasma levels ofdesmosine/isodesmosine; C = Caucasian; H = Hispanic. *Patient 1 hashomozygous ZZ α₁-antitrypsin deficiency.Elastase Digestion

To determine EDPs produced by elastases in human lung, we digested humanlung elastin by 2 representative elastolytic enzymes, human neutrophilelastase (HNE) isolated from human sputum¹⁰⁸ and macrophagemetalloproteinase (MMP12) isolated from mouse peritoneal lavage¹⁰⁹,which has been shown to be a mouse orthologue of human alveolarmacrophages metalloproteinase^(110,111).

Human lung elastin (4 mg) was suspended in 1 mL of 0.1 M ammoniumcarbonate buffer, pH 7.8. The suspension was digested by addition of 0.1mg HNE (875 U/mg) or 25 μg MMP12 (37.5 U/mg) or 7 mg PPE (8 U/mg) andstirring at room temperature for 12 hours. After 12 hours the digestionswere repeated for the second time by addition of another portion offresh enzymes. The digested mixtures were fractionated into 3 fractionsof molecular weight cut-off: (1)<10,000, (2) 10,000-50,000, (3) >50,000Da using Centricon (Millipore, Mass.) membrane filtration tubes.

Characterization of Peptides by LC/MSMS Analysis

A TSQ Discovery electrospray tandem mass spectrometer (Thermo Electron)was used for both LC/MS and LC/MSMS analysis. High-performance liquidchromatography (HPLC) conditions involved the use of a Symmetry 1 mm×5cm dC18 (5 μm) column (Waters, Milford, Mass.) and programming from a 5%acetonitrile/water (0.1% formic acid) to 50% acetonitrile/water (0.1%formic acid) for 50 minutes under a flow rate of 100 μL/min. LC/MSanalysis was carried out in positive ion mode with a spray voltage of4,000 volts and an ion transfer tube temperature of 300° C. LC/MSMSanalysis was performed by stepping up collision energy from 20 to 40 eVwith the increase in the molecular weights from 500 to 1500 Da. Thepeptide sequences were assigned by searching their MS/MS spectra againstthe SwissProt database by either PEAK (Bioinformatics Solution,Waterloo, Canada) or MASCOT (Matrix Science, MA) software.

Selected Reaction Monitoring (SRM) of EDPs in Body Fluids

Plasma samples were obtained after centrifuging venous blood specimensat 2500 revolutions per minute for 25 minutes. Samples were stored at−20° C. until used. Sputum samples of COPD patients were collected fromspontaneously produced sputum. All patients gave informed consent forthe study, and the study was approved by the institutional review board.

One milliliter of plasma or sputum from selected COPD patients werefiltered by a Centricon membrane to obtain molecular weight cut-offfractions of <10,000 Da. One transition ion (the most abundant CID ion)was selected from each characterized EDP, and they were searched for thepresence of the corresponding EDPs in the LC/MSMS spectra obtained frombody fluids of COPD patients. Four synthetic peptides, GYPI (SEQ IDNO:5), APGVGV (SEQ ID NO:4), GLGAFPA (SEQ ID NO:11), and VGVLPGVPT (SEQID NO:16), were used for structural confirmation and as externalstandards for the SRM quantitation.

Results

Identification of Lung Elastin-Derived Peptides (EDPs) Produced byNeutrophil and Macrophage Metalloproteinase Digestions

Samples of human lung elastin were digested by HNE and MMP12. Thedigested peptide mixtures were separated into 3 molecular weight cut-offfractions: (1) smaller than 10,000, (2) between 10,000 and 50,000, and(3) larger than 50,000 Da. LC/MS analysis of the peptide fractionsindicated that a large portion of soluble EDPs was isolated in fraction1 (<10,000 Da). Fraction 2 (10,000-50,000 Da) contained essentially thesame peptides as that of the fraction 1 but in significantly lowerconcentration. Fraction 3 (>50,000 Da) contained mostly the undigestedsolid elastin. Therefore, fraction 1 (<10,000 Da) appears to constituteall major soluble EDPs produced by HNE or MMP12 digestion of lungelastin, as shown in FIGS. 26A and 26B. Each of the major ions in theLC/MS chromatogram of FIGS. 26A and 26B were further fragmented by thetandem mass spectrometry (LC/MSMS) analysis to obtain collision induceddissociation (CID) ion spectra that provide information from which aminoacid sequences of each peptide was derived^(112,113). We utilized acomputer program, PEAK, to assist de novo interpretation of the CID ionspectra and deduced their amino acid sequences. MASCOT and manualassignments were also utilized to achieve unambiguous assignments, whichwere searched against the database of human tropoelastin sequences inSwissProt database (P15502) to characterize the EDPs. By this approach,we were able to characterize all of the major peptides, i.e., 24 EDPsproduced by the HNE digestion (Table 7) and 16 EDPs produced by theMMP12 digestion (Table 8). FIG. 27 shows an example of the amino acidsequence assignment and characterization of peptide VGVLPGVPT (SEQ IDNO:16).

TABLE 7  Human Lung EDPs (<10,000 da) by HNE Digestion Positions in HPLCLC/MS Amino acid tropoelastin Peptides (min) (m/z) sequence (P15002) 12.2 444 LGVGV 582-586 2 3.4 444 GGIPT 392-386 3 3.7 499 APGVGV 509-514515-520 527-532 533-538 539-544 4 12.3 449 GYPI 205-208 5 13.3 520 VTFPG85-89 6 15.5 598 GIPGGVV 675-681 7 16.0 866 APGIGPGGVAA 545-555 8 17.7598 GVPGLGV 587-593 596-605 9 18.0 572 GLGGLGV 708-714 10 18.1 632GLGAFPA 78-84 11 18.1 763 GGIPTYGV 392-399 12 18.9 726 GAGVPGLGV 594-60213 19.3 643 AGLGGLGV 707-714 14 20.1 771 GAAGLGGLGV 705-714 15 21.5 838VGVLPGVPT 163-171 16 21.6 754 GVGVPGLGV 585-593 17 22.1  543*AARPGFGLSPI 760-770 18 23.9 930 GLGAGLGAFPA 74-84 19 24.4 865 GAGGFPGFGV400-409 20 26.4 1209  GLVPGGPGFGPGVV 321-334 21 26.9  779*GVPGAGVPGVGVPG 335-353 AGIPV 22 27.8 1139  FPGVGVLPGVPT 160-171 23 39.8 731* GVGVPGLGVGAGVP 585-602 GLGV 24 30.8  728* GLGGVLGGAGQFPL 743-759GGV *Doubly charged ion.Peptides 1-24 in Table 7 above correspond to SEQ ID NOS:2-25).

TABLE 8 Human Lung EDPs (<10,000 da) by MMP12 DigestionTABLE 4 Human Lung EDPs (<10,000 da) by MMP12 Digestion AminoPositions In Positions in HPLC LC/MS acid tropoelastin HPLC LC/MSAmino acid tropoelastin Peptides (min) (m/z) sequence (P5002) Peptides(min) (m/z) sequence (P5002) 1 1.5 499 VAPGVG 506-511 9 11.2 476 FPGVG160-164 512-517 518-523 530-535 536-541 542-547 2 2.2 556 VGAGVPG593-599 10 13.5 726 LGVGAGVPG 591-599 602-608 600-608 3 2.7 513 LAPGVG526-531 11 15.1 591 VTFPGA 85-90 4 3.5 596 LVPGGPG 322-327 12 15.2 575LGAFPA 79-84 5 7.5 655 VGVAPGVG 506-513 13 17.0  543* VYPGGVLPGAR149-159 512-519 519-525 530-537 536-543 6 8.5 584 VGVGVPG 584-590 1418.2 858 VYPGGVLPG 149-157 7 9.5 541 LVPGVG 502-507 15 21.0 859FGVGVGGIP 407-416 8 10.7 584 LGAGPG 575-581 16 23.7 925 LGVGVGVPGLG582-592 *Doubly charged ion.Peptides 1-16 in Table 8 above correspond to SEQ ID NOS:26-41).

The peptides are rich in nonpolar amino acids, especially G, V, P, A, L,or I, including 2 hexapeptides, APGVGV (SEQ ID NO:4) and VAPGVG (SEQ IDNO:26), and an octapeptide, VGVAPGVG (SEQ ID NO:30), derived from thecharacteristic elastic repeats (positions 506 to 547; see Table 5) ofthe hydrophobic domain in tropoelastin^(84,114,115).

Detection of Lung EDPs in Body Fluids of COPD

Using the SRM of LC/MSMS analysis (see Materials and Methods), thetransition ions derived from the 24 EDPs characteristically produced byHNE digestion and the 16 EDPs characteristically produced by MMP12digestion were used to search for their presence in plasma or sputumobtained from COPD patients. Five patients were selected at random fromsubjects diagnosed with COPD. The results showed that 3 or 4 peptides,GYPI (NO: 5), APGVGV (SEQ ID NO:4), GLGAFPA (SEQ ID NO:11), andVGVLPGVPT (SEQ ID NO:16), were present in plasma or sputum from 2 COPDpatients (patients 1 and 2), but not in 3 other COPD patients and 4normal controls (Table 9). The identities and quantities of the detectedpeptides were determined by comparison with the synthetic peptides. TheSRM is highly sensitive and specific with a limit of detection, 0.01 ngof peptide present in 1 mL of plasma. FIG. 28 describes theidentification and quantification of the peptide in plasma of COPDpatient 1.

TABLE 9  Lung EDPs Detected in COPD Patients GYPI APGVGV GLGAFPAVGVLPGVPT Patients 1 Plasma 0.14 0.11 0.48 0.10 Sputum X* X 1.01 X 2Plasma 0.02 X 1.44 0.04 Sputum X X X X 3 Plasma X X X X 4 Plasma X X X X5 Plasma X X X X Normal subjects 1 Plasma X X X X 2 Plasma X X X X 3Plasma X X X X 4 Plasma X X X XHexapeptides APGVGV, VAPGVG, and VGVAPG from the Elastin Repeats

Several EDPs such as the hexapeptide VGVAPG (SEQ ID NO:42), which hasbeen isolated from bovine ligament elastin by porcine pancreaticelastase (PPE) digestion, have been actively studied as chemoattractantsfor neutrophils and macrophages¹¹⁶. However, our LC/MS studies show thatthe hexapeptide APGVGV (SEQ ID NO:4) is formed by HNE and VAPGVG (SEQ IDNO:26) by MMP12 digestion, with no detection of VGVAPG. We have extendedour LC/MSMS analysis to EDPs obtained with the PPE digestions on bothlung elastin and bovine ligament elastin. The PPE digestions resulted incharacterization of 12 EDPs from Lung elastin (Table 10) and 21 EDPsfrom ligament elastin (Table 11), and it was found that VGVAPG wasexclusively formed by PPE digestion or from digestion of ligamentelastin (FIG. 29) but not from digestion with neutrophil or macrophagemetalloproteinase. Three additional hexapeptides, GGLVPG (SEQ ID NO:57),VGPGLG (SEQ ID NO:75), and PGLGVG (SEQ ID NO:62), were also identifiedin the bovine ligament digests by PPE. All 5 hexapeptides are isobaric(the same molecular weight, 499 Da), but their amino acid sequences arevaried and can be defined by LC/MSMS.

TABLE 10 Human Lung EDPs (<10,000 Da) by PPE Digestion Positions in HPLCLC/MS Amino acid tropoelastin Peptides (min) (m/z) sequence (P15002) 12.6 499 VGVAPG 506-511 512-517 518-523 530-535 536-541 542-547 2 4.5 442VGLPG 144-148 3 8.4 456 AGIPV 349-353 4 9.4 449 GYPI 205-208 5 10.7 419VGPF 192-195 6 16.2 912 VGPFGGLPQPG 192-201 7 18.0 563 QFGLV 499-503700-704 8 18.6 786 GPGFGPGVV 326-334 9 21.6 633 FGLSPI 765-770 10 21.61011 GLVPGGPGFGPG 321-332 11 24.0 737 GGFPGFGV 402-409 12 26.1 1209GLVPGGPGFGPGVV 321-334Peptides 1-12 in Table 10 correspond to SEQ ID NOS:42-53.

TABLE 11 Bovine Ligament EDPs (<10,000 Da)  by PPE DigestionPositions in HPLC LC/MS Amino acid tropoelastin Peptides (min) (m/z)sequence (P04985) 1 2.4 702 GGVGDLGGA 619-627 2 2.6 499 VGVAPG 503-508521-526 3 3.9 442 VGLPG 153-157 4 5.8 449 GGLVPG 5 7.6 473 AGLGGV692-697 6 9.0 449 GYPI 216-219 7 10.3 419 VGPF 203-206 8 11.5 681PGVGVVPG 507-514 513-520 9 13.0 499 PGLGVG 566-571 575-580 584-589 1013.4 852 GGQQPGVPL 207-215 11 14.5 490 FPGIG 171-175 429-433 12 16.7 433FPGI 171-174 FPGI 429-432 13 17.9 591 GGIPTF 417-422 14 17.9 804GFPGIGDAA 428-436 15 19.6 658 GQPFPI 701-706 16 20.9 1139 VGPFGGQQPGVP203-214 17 22.5 845 FPGAGLGGLG 43-52 18 24.8 856 FPGIGVLPG 171-179 1924.8 1252 VGPFGGQQPGVPL 203-215 20 26.5 751 GVFFPGAG 40-47 21 26.5 694VFFPGA 41-47Peptides 1-21 in Table 11 correspond to SEQ ID NOS:54-74.Discussion

In this study enzymatic digestion of human lung elastin by 2 keyenzymes, human neutrophil elastase (HNE) and macrophagemetalloproteinase (MMP12), resulted in identification of 24 EDPs fromHNE (Table 7) and 16 EDPs from MMP12 (Table 8). The 40 EDPs we havecharacterized by LC/MSMS analysis appear to represent all major solubleEDPs produced by the HNE and MMP12 digestions of elastin in vitro. Thepeptides are rich in nonpolar amino acids, especially G, V, P, A, L, orI, from the hydrophobic elastic domain in tropoelastin. Among them, thehexapeptides APGVGV (SEQ ID NO:4), and VAPGVG (SEQ ID NO:26) and theoctapeptide VGVAPGVG (SEQ ID NO:30) are derived from the characteristichydrophobic elastic repeats (positions 506 to 547; Table 5).Tropoelastin consists of alternating repetitive hydrophobic domains ofvariable length (the elastic repeats) and the alanine-richlysine-containing domain that form cross-links^(114,115). A model of theelastic property of the elastin matrix proposes that hydrophobicinteractions of the 3-spiral elastic repeats are exposed to an aqueousenvironment to stabilize the folded protein structure¹¹⁷⁻¹¹⁹. Thespectrum of EDPs we have characterized suggests that both HNE and MMP12degradations occur primarily at the site of the hydrophobic elasticrepeat domain and result in disintegration of the stable proteinstructure of elastin.

Recent progress in proteomic LC/MSMS analysis has become an effectiveapproach to characterize protein molecules as biomarkers involved indisease. This study represents the first effort to determine peptidefragments from enzymatic degradations in biological fluids in COPDpatients. The 40 EDPs we have characterized by LC/MSMS analysis resultsfrom the 2 major elastases involved in elastin degradation in COPD. Webelieve these EDPs represent all the major soluble EDPs produced by HNEand MMP12 digestions. However, the amino acid sequences produced in ourstudy differ from the sequences produced in the study reported byBarroso et al.¹⁰⁰. This may be a consequence of a difference in theenzymes used or a difference in the conditions of digestion.

We used the 40 characterized EDPs to search for biomarkers of lungelastin degradation in body fluids obtained from COPD patients. Ourpreliminary screening of some selected COPD patients have shown that 4EDPs, GYPI (SEQ ID NO: 5), APGVGV (SEQ ID NO:4), GLGAFPA (SEQ ID NO:11),and VGVLPGVPT (SEQ ID NO:16), were present in plasma or sputum of 2 COPDpatients but not in 3 other COPD subjects or healthy controls (Table 9).Two patients (patients 1 and 2) with detectable levels of EDPs have moreadvanced disease clinically and physiologically. Patient 1 with AATD hadevidence of emphysematous destruction on chest x-ray and computedtomography (CT) as did patient 2. The 3 other subjects with undetectablelevels of EDPs had mild or little evidence of emphysematous destructionon chest x-ray or CT. All of the detected peptides were among thoseidentified from HNE digestions in vitro, suggesting that this was theactive enzyme producing the elastin degradation in vivo in thesepatients, an observation especially relevant to the patients with AATD.Further study is needed to determine whether the presence of multiplepeptides as shown here can be related to the severity of emphysematousdestruction by CT. Such studies are planned along with furtherevaluation of the chemotactic and antigenic potential of the detectedpeptides. The possible reasons for not detecting EDPs in all patientsinclude (1) levels too low for detection by our mass spectrometryanalysis; (2) in vivo intermittent production of such peptides dependingupon varying pathological factors; and (3) varying phenotypes of diseasein COPD related to causative factors and the host responses. Furtherstudy in COPD patients may clarify the basis of these variable results.

The EDPs represented by the hexapeptide VGVAPG (SEQ ID NO:42) isolatedfrom ligamentum nuchae or aorta of animals by porcine pancreaticelastase (PPE) have been shown to be chemoattractants for neutrophilsand macrophages, and thus initiators of possible pathogenic consequencesof elastin degradation^(102-105,120). Also, VGVAPG has been reported toinduce pro-MMP1 and pro-MMP3 upregulation¹²¹. However, our study withhuman elastin showed that the hexapeptides APGVGV (SEQ ID NO: 4) andVAPGVG (SEQ ID NO:26) were formed by degradation with human neutrophilelastase (HNE) and macrophage metalloproteinase (MMP12), The hexapeptideVGVAPG (SEQ ID NO:42) could not be detected by the digestion of lungelastin by either HNE or MMP12. VGVAPG (SEQ ID NO:42) was only formedeither by the PPE digestion or formed by the digestion of ligamentumnuchae (FIG. 29), and may not be a factor in COPD. Further studies onbiological activities of these hexapeptides such as their chemotactic orantigenic properties in relation to the pathogenesis of COPD are underway.

In sum, we utilized proteomic LC/MSMS analysis to characterize the fullspectrum of peptides that can be produced by 2 major elastases(neutrophil elastase and macrophage metalloproteinase) in vitro fromhuman lung elastin. These characterized elastin peptides were searchedfor and several were detected in plasma and sputum in COPD patients.Such elastin peptides were not detected in normal subjects. Thedetection in vivo of these elastin peptides in COPD patients appears tobe varied, which might be a reflection of variable pathogenic processesin COPD. This study demonstrates the feasibility of detecting theseelastin peptides in body fluids and relating them to clinical,physiological, and radiological characteristics of COPD and makespossible further study for their pathogenic potential.

All documents cited herein are hereby incorporated by reference as ifrecited in full herein.

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Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

What is claimed is:
 1. A method for identifying candidate compounds thatare effective to treat, prevent, or ameliorate the effects of a diseasecharacterized by elastin degradation comprising: (a) administering acandidate compound to a cell culture model of the disease; (b)measuring, by mass spectrometry using an internal standard comprisingacylated pyridinoline, the amount of at least one marker of elastindegradation in the cell culture administered the candidate compound,wherein the at least one marker of elastin degradation is selected fromthe group consisting of desmosine, isodesmosine, and combinationsthereof; and (c) determining whether the amount of the at least onemarker produced by the cell culture administered the candidate compoundis different compared to a control cell culture absent the candidatecompound, wherein a decrease in the amount of the at least one markerproduced by the cell culture administered the candidate compoundcompared to the control cell culture identifies the candidate compoundas effective to treat, prevent, or ameliorate the effects of thedisease, wherein the disease is selected from the group consisting ofchronic obstructive pulmonary disease (COPD), COPD with AATD, chronicbronchitis, emphysema, and refractory asthma.
 2. The method according toclaim 1, wherein the disease is COPD.
 3. The method according to claim1, wherein both desmosine and isodesmosine are measured.
 4. The methodaccording to claim 1, wherein the candidate compound is selected fromthe group consisting of hyaluronic acid, polysaccharide, carbohydrate,small molecules, and RNAi.
 5. The method according to claim 1, whereintandem mass spectrometry is used.
 6. A method for identifying candidatecompounds that are effective to treat, prevent, or ameliorate theeffects of a disease characterized by elastin degradation comprising:(a) administering a candidate compound to a cell culture model of thedisease; (b) measuring, by mass spectrometry using an internal standardcomprising acylated pyridinoline, the amount of desmosine andisodesmosine in the cell culture administered the candidate compound;and (c) determining whether the amount of desmosine and isodesmosineproduced by the cell culture administered the candidate compound isdifferent compared to a control cell culture absent the candidatecompound, wherein a decrease in the amount of the desmosine andisodesmosine produced by the cell culture administered the candidatecompound compared to the control cell culture identifies the candidatecompound as effective to treat, prevent, or ameliorate the effects ofthe disease, wherein the disease is selected from the group consistingof chronic obstructive pulmonary disease (COPD), COPD with AATD, chronicbronchitis, emphysema, and refractory asthma.
 7. The method according toclaim 6, wherein tandem mass spectrometry is used.